Addition Of Auxin Research Articles

Effects of the auxin-dependent degradation of the cohesin and condensin complexes on the repair of distant DNA double-strand breaks in mouse embryonic stem cells.

The SMC protein family, including cohesin and condensin I/II, plays a pivotal role in maintaining the topological structure of chromosomes and influences many cellular processes, notably the repair of double-stranded DNA breaks (DSBs). The cohesin complex impacts DSB repair by spreading γH2AX signal and containing DNA ends in close proximity by loop extrusion. Cohesin supports DNA stability by sister chromatid cohesion during the S/G2 phase, which limits DNA end mobility. Cohesin knockdown was recently shown to stimulate frequencies of genomic deletions produced by distant paired DSBs, but does not affect DNA repair of a single or close DSBs. We examined how auxin-inducible protein degradation of Rad21 (cohesin) or Smc2 (condensins I+II) changes the frequencies of rearrangements between paired distant DSBs in mouse embryonic stem cells (mESCs). We used Cas9 RNP nucleofection to generate deletions and inversions with high efficiency without additional selection. We determined optimal Neon settings and deletion appearance timings. Two strategies for auxin addition were tested (4 independent experiments in total). We examined deletion/inversion frequencies for two regions spanning 3.5 and 3.9 kbp in size. Contrary to expectations, in our setting, Rad21 depletion did not increase deletion/inversion frequencies, not even for the region with an active Ctcf boundary. We actually observed a 12 % decrease in deletions (but not inversions). At the same time, double condensin depletion (Smc2 degron line) demonstrated high biological variability between experiments, complicating the analysis, and requires additional examination in the future. TIDE analysis revealed that editing frequency was consistent (30-50 %) for most experiments with a minor decrease after auxin addition. In the end, we discuss the Neon/ddPCR method for deletion generation and detection in mESCs.

Pros and cons of auxin-inducible degron as a tool for regulated depletion of telomeric proteins from Saccharomyces cerevisiae.

To assess the immediate responses of the yeast cells to telomere defects, we employed the auxin-inducible degron (AID) enabling rapid depletion of essential (Rap1, Tbf1, Cdc13, Stn1) and non-essential (Est1, Est2, Est3) telomeric proteins. Using two variants of AID systems, we show that most of the studied proteins are depleted within 10-30 min after the addition of auxin. As expected, depletion of essential proteins yields nondividing cells, provided that the strains are cultivated in an appropriate carbon source and at temperatures lower than 28°C. Cells with depleted Cdc13 and Stn1 exhibit extension of the single-stranded overhang as early as 3 h after addition of auxin. Notably, prolonged incubation of strains carrying AID-tagged essential proteins in the presence of auxin resulted in the appearance of auxin-resistant clones, caused at least in part by mutations within the OsTIR1 gene. Upon assessing the length of telomeres in strains carrying AID-tagged non-essential telomeric proteins, we found that the depletion of Est1 and Est3 leads to auxin-dependent telomere shortening. However, the EST3-AID strain had slightly shorter telomeres even in the absence of auxin. Furthermore, a strain with the AID-tagged version of Est2 (catalytic subunit of telomerase) not only had shorter telomeres in the absence of auxin but also did not exhibit auxin-dependent telomere shortening. Our results demonstrate that while AID can be useful in assessing immediate cellular responses to telomere deprotection, each strain must be carefully evaluated for the effect of AID-tag on the properties of the protein of interest.

The Effect of NAA, BAP and NAA, BAP Combinations on the Growth of In-vitro Culturally Promoted Leaves of Sansevieria ehrenbergii

Sansevieria is an ornamental succulent leaf plant of the family Agavaceae, having a beautiful and diverse shape and color. Propagation of Sansevieria can be done by tissue culture method with the addition of cytokinins and auxins to MS media. The study used a complete randomized design consisting of 8 treatments, 3 tests, there were 24 experimental units. The observed parameters are when the callus appears, the color of the callus, the texture of the callus, the weight of the callus, the diameter of the callus, and the percentage of success in growing. Data analysis used variance analysis and tested using 5% DNMRT. The data is processed using a Statistical Analysis System (SAS). The results showed that the application of NAA, BAP, and the combination of NAA and BAP affected the per-plant explant of Sansevieria ehrenbergii leaves on all observation parameters. The treatment of NAA 2 mg.l-1 + BAP 1 mg.l-1 gave effect tending to be faster when callus appeared with an average of 75.75 HST, NAA 1 mg.l-1 + BAP 2 mg.l-1 m. and tends to be larger in callus diameter with an average of 0.9033 g and 19.133 mm and NAA 1 mg.l-1 + BAP 1 mg.l-1 gives a higher influence on the percentage of callus growth by an average of 100 %.

Callusogenesis and Organogenesis of Thymus Cultivars In Vitro

The article is devoted to the in vitro cultivation of three Russian cultivars of two Thymus species (Th. vulgaris and Th. serpyllum) in order to study the possibility of induction of callusogenesis and organogenesis. The developed technology opens up prospects for the induction of somaclonal variability, obtaining the source material for breeding, as well as regeneration of thyme plants after genetic transformation. For the study, we used nutrient media based on the mineral composition of the Murashige and Skoog medium with the addition of auxin (indole-3-butyric acid, indole-3-acetic acid) and cytokinin (6-benzyladenine, kinetin) substances. For the induction of callusogenesis, the use of stem explants on nutrient media supplemented with 2,4-dichlorphenoxyacetic acid (1 or 2 mg/L) can be recommended. The efficiency of shoot organogenesis also depends on the type of explant, and its rather low values in most experimental variants are apparently due to the accumulation of endogenous auxins and phenolic compounds. Thus, a cultivar-specific morphogenetic response to the use of various phytohormones and growth regulators was established, as well as the influence of the explant type on the efficiency of morphogenic processes.

Methodology to Create Auxin-Inducible Degron Tagging System to Control Expression of a Target Protein in Mammalian Cell Lines.

The auxin-inducible degron (AID) system is a versatile tool in cell biology and genetics, enabling conditional protein regulation through auxin-induced degradation. Integrating CRISPR/Cas9 with AID expedites tagging and depletion of a required protein in human and mouse cells. The mechanism of AID involves interactions between receptors like TIR1 and the AID tag fused to the target protein. The presence of auxin triggers protein ubiquitination, leading to proteasome-mediated degradation. We have used AID to explore the mitotic functions of the replication licensing protein CDT1. Swift CDT1 degradation via AID upon auxin addition achieves precise mitotic inhibition, revealing defects in mitotic spindle structure and chromosome misalignment. Using live imaging, we found that mitosis-specific degradation of CDT1 delayed progression and chromosome mis-segregation. AID-mediated CDT1 inhibition surpasses siRNA-based methods, offering a robust approach to probe CDT1's mitotic roles. The advantages of AID include targeted degradation and temporal control, facilitating rapid induction and reversal of degradation-contrasting siRNA's delayed RNA degradation and protein turnover. In summary, the AID technique enhances precision, control, and efficiency in studying protein function and regulation across diverse cellular contexts. In this article, we provide a step-by-step methodology for generating an efficient AID-tagging system, keeping in mind the important considerations that need to be adopted to use it for investigating or characterizing protein function in a temporally controlled manner. Key features • The auxin-inducible degron (AID) system serves as a versatile tool, enabling conditional protein regulation through auxin-induced degradation in cell biology and genetics. • Integration of CRISPR/Cas9 knock-in technology with AID expedites the tagging and depletion of essential proteins in mammalian cells. • AID's application extends to exploring the mitotic functions of the replication licensing protein CDT1, achieving precise mitotic inhibition and revealing spindle defects and chromosome misalignment. • The AID system and its diverse applications advance the understanding of protein function and cellular processes, contributing to the study of protein regulation and function.

Mechanistic insights into auxin-enhancing polycyclic aromatic hydrocarbon uptake by wheat roots: Evidence from in situ intracellular pH and root-surface H+ flux

Polycyclic aromatic hydrocarbons (PAHs) are a group of extremely carcinogenic organic pollutants. Our previous findings have demonstrated that plant roots actively take up PAHs through co-transport with H+ ions. Auxin serves as a pivotal regulator of plant growth and development. However, it remains unclear whether the hormone can enhance the uptake of PAHs by plant roots. Hence, the wheat root exposed to PAHs with/without auxins was set to investigate how the auxin promotes the PAHs uptake by roots. In our study, auxin could significantly enhance the uptake of PAHs after 4 h of exposure. After the addition of auxin, the root tissue cytoplasmic pH value was decreased and the H+ influx was observed, indicating that the extracellular space was alkalinized in a short time. The increased H+ influx rate enhanced the uptake of PAHs. In addition, the H+-ATPase activity was also increased, suggesting that auxin activated two distinct and antagonistic H+ flux pathways, and the H+ influx pathway was dominant. Our findings offer important information for exploring the mechanism underlying auxin regulation of PAHs uptake and the phytoremediation of PAH-contaminated soil and water.

The effect of plant growth regulators on micropropagation of Melientha suavis Pierre. and assessment of genetic fidelity of regenerants based on iPBS and SRAP markers

Abstract. Siringam T, Vanijajiva O. 2023. The effect of plant growth regulators on micropropagation of Melientha suavis Pierre. and assessment of genetic fidelity of regenerants based on iPBS and SRAP markers. Biodiversitas 24: 4628-4634. Melientha suavis Pierre, a significant deciduous edible plant species with high nutritional value and belonging to the Opiliaceae family, holds paramount importance for both agricultural and conservation purposes in its native Southeast Asia. However, the species faces challenges due to habitat fragmentation and negative anthropogenic impacts, leading to a decline in its population. This study aimed to explore the effects of various concentrations of plant growth regulators (cytokinin and auxin) on the shoot and root development of M. suavis, and to assess the genetic stability of micropropagated plants obtained from nodal explants. In vitro shoot regeneration and proliferation were conducted on Murashige and Skoog (MS) semi-solid medium, supplemented with 6-benzylaminopurine (BAP) or kinetin (Kn) at different doses. The optimal shoot length, shoot number, and leaf volume were observed in the modified MS medium with 1.0 mg/L BAP after an 8-week incubation period. Efforts to enhance the rooting of micropropagated shoots included the addition of auxins, such as ?-naphthalene acetic acid (NAA) or indole-3-butyric acid (IBA). However, the species exhibited recalcitrant behavior during the reproduction and rooting stages, as the rooting percentage did not correlate with the increase in auxin concentration. Interestingly, the highest root number and length were achieved in the MS medium without plant growth regulators after 8 weeks of incubation. To ensure genetic fidelity, regenerants were subjected to inter-primer binding site (iPBS) and sequence-related amplified polymorphism (SRAP) marker analysis. The results revealed no genetic variation between the micropropagated plants and the mother trees, confirming the production of genetically stable progeny. Overall, this protocol presents a promising alternative for profitable propagation and establishment of genetically constant progeny of M. suavis, with important implications for sustainable applications and germplasm conservation in Southeast Asia. Addressing the challenges faced by this valuable species through micropropagation and genetic fidelity assessment can contribute significantly to its preservation and utilization for agricultural and conservation initiatives.

Indirect organogenesis of Aceh patchouli leaf explants (Pogostemon cablin Benth) by in vitro

The Aceh patchouli plant (Pogostemon cablin. Benth) is a plantation plant that produces essential oils with great potential, known as patchouli oil. The use of in vitro culture techniques on patchouli plants needs to be developed to meet the needs of patchouli seeds. This study aimed to analyze the effect of plant growth regulators (PGRs) cytokinins 6-Benzyl Amino Purine (BAP) and Thidiazuron (TDZ) and auxin 1-Naphthaleneacetic Acid (NAA) on the organogenesis ability of Aceh patchouli leaf explants var. Lhokseumawe. The treatments analyzed were P1 = MS; P2 = BAP 0.75 mg.l−1, P3 = BAP 1.0 mg.l−1, P4 = TDZ 0.75 mg.l−1, P5 = TDZ 1.0 mg.l−1, P6 = NAA 0.5 mg.l−1, P7 = NAA 1.0 mg.l−1, P8 = BAP 0.75 mg.l−1 + NAA 0.5 mg.l−1, P9 = BAP 0.75 mg.l−1 + NAA 1.0 mg.l−1, P10 = BAP 1.0 mg.l−1 + NAA 0.5 mg.l−1, P11 = BAP 1.0 mg.l−1 + NAA 1.0 mg.l−1, P12 = TDZ 0.75 mg.l−1 + NAA 0.5 mg.l−1, P13 = TDZ 0.75 mg.l−1 + NAA 1.0 mg. l−1, P14 = TDZ 1.0 mg.l−1 + NAA 0.5 mg.l−1, P15 = TDZ 1.0 mg.l−1 + NAA 1.0 mg.l-1. The percentage of callus induction observed, percentage of shoot induction, percentage of browning, percentage of contamination and percentage of live explants at 28 DAI. The results showed that the percentage of explants forming callus was 56.4%, and the percentage of explants forming organ was 54.6%. In these two parameters the treatment of P14 and P15 gave the best results of 6.6% respectively. The percentage of explants experiencing browning was 26.1%, treatment P1 gave the highest response for this parameter, namely 6.6%. The parameter percentage of live explants was 71.3%. The best response was found in treatment P2, P3, P12 and P15, which were 6.6% each. The fastest bud formation occurred at 14 Days After Initiation (DAI) in P4 treatment. The resulting shoots look quite vigor and have formed leaves. In the combination treatment P14, showed the best results, the addition of auxin was able to increase the initiation of embryogenic callus.

Somatic embryogenesis as potential method for commercial propagation in Passiflora edulis Sims f. edulis - an important horticultural crop

Purple passion fruit (Passiflora edulis Sims f. edulis) is one of the species with high commercial value in the genus Passiflora. In this study, the somatic embryogenesis (SEs) obtained from different explants (ex vitro leaf and in vitro root) cultured on medium-supplemented auxins and cytokinins, silver nanoparticles (AgNPs) enhanced SEs and plantlets derived from SEs were evaluated for the first time. The ex vitro leaf blade (1 × 1 cm) and in vitro root fragment (0.1 × 1 cm) were used as the initial explants. The results showed that the addition of auxins and cytokinins in culture medium stimulated SEs in both explant types. Root explant only induced callus on medium supplemented with 2,4-D (1.0 - 4.0 mg L−1), however, somatic embryos were directly induced with 1.0 mg L−1 NAA. While 1.0 - 2.0 mg L−1 2,4-D stimulated embryogenic callus induction (14.81 - 25.93%), NAA stimulated direct embryogenesis from leaf explants. The medium supplemented with 2.0 mg L−1 NAA combined with 1.5 mg L−1 TDZ significantly improved the SEs rate (81.48%) and number of embryos per explant (26.67 embryos) formed directly from leaf explants. On the other hand, the synergism between 2.0 mg L−1 2,4-D and 2.0 mg L−1 AgNPs significantly enhanced callus-derived embryogenesis from leaf explant with the highest SEs rate (92.59%) and number of embryos per explant (31.67 embryos). In addition, AgNPs also had a positive effect on promoting the maturation of callus-derived embryos. The plantlets derived from SEs were acclimatized in the greenhouse with a survival rate of over 90.00%. The SEs-derived plants flowered and produced fruit normally under field conditions. The results revealed a reliable plant regeneration procedure through direct or indirect SEs derived from ex vitro leaf and in vitro root explants and AgNPs-stimulated SEs that can be applied for commercial propagation of this crop.

Effects of the addition of phytohormone and plant growth-promoting bacteria on the health and development of Polygonum hydropiperoides cultivated in constructed wetlands treating chromium-contaminated wastewater

Constructed wetland (CW) systems are used for treating different types of wastewaters, including tannery effluent, which are rich in heavy metals. The purpose of this study was to assess the potential use of Polygonum hydropiperoides and its chromium removal efficiency in CWs treating synthetic tannery wastewater (STW), after the addition of phytohormone auxin and plant growth-promoting bacteria (PGPB) Azospirillum brasiliense. We installed and operated nine pilot-scale CWs named as: CWP+B – unit with phytohormone and PGPB; CWP – unit with phytohormone; CWB – unit with PGPB; CWV – vegetated unit, without PGPB and auxin, and CWUV – unvegetated unit. Also, each vegetated CW had a parallel control unit to evaluate plant phytotoxicity. According to the results, all CWs were efficient in chromium removal and there were no significant differences among them. The visual symptoms observed in the aerial parts of the individuals exposed to chromium-rich wastewater were curling, necrosis, chlorosis and senescence of the leaves. The most intense positive detection of cell death occurred in the leaves and roots of the individuals exposed to chromium. In the vegetated wetlands with added phytohormone and PGPB, there were fewer visual damages, lower detection of cell death in the leaves and roots, and an increase in growth and productivity of P. hydropiperoides.

Auxin alone provokes retention of ASH1 mRNA in Saccharomyces cerevisiae mother cells.

The auxin-inducible degradation (AID) system can elicit conditional and reversible protein degradation as a tool to assess the role of essential proteins. Indeed, AID enables functional studies without the possibility of adaptation, which can occur with permanent gene deletions. The AID system relies on the addition of auxin molecules, such as indole-3-acetic acid (IAA), as a means to launch the degradation of the protein of interest. In this context, it is extremely important to control for the effect of auxin addition alone. To study the role of essential proteins in the process of selective mRNA delivery to daughter cells in Saccharomyces cerevisiae , we first controlled for the effect of adding IAA to cells that cannot perform AID-mediated degradation. We found that auxin alone restricted ASH1 delivery to daughter cells, as ASH1 mRNA started being retained in the mother cell as soon as thirty minutes after IAA addition. Thus, our data warn about the danger of not systematically including auxin-treated cells incapable of degradation in every AID-related experiment. Furthermore, given previous data reporting the ability of auxin to inhibit the master growth regulator TORC1 in S. cerevisiae , our data suggest that TORC1 could control the selective delivery of mRNAs to daughter cells.

Prediction of In vitro organogenesis of Bacopa monnieri using artificial neural networks and regression models

This study was conducted to determine if artificial neural networks (ANN) can be used to accurately predict in vitro organogenesis of Bacopa monnieri compared with statistical regression. Prediction models were developed for shoot and root organogenesis (outputs) on two culture media (Murashige and Skoog and Gamborg B5) affected by two explant types (leaf and node) and two cytokinins (6-Benzylaminopurine and Thidiazuron at 1.0, 5.0, and 10.0 μM levels) with and without the addition of auxin (1-Naphthaleneacetic acid 0.1 μM) (inputs). Categorical data were encoded in numeric form using one-hot encoding technique. Backpropagation (BP) and Kalman filter (KF) learning algorithms were used to develop nonparametric models between inputs and outputs. Correlations between predicted and observed outputs (validation dataset) were similar in both ANN-BP (R values = 0.77, 0.71, 0.68, and 0.48), and ANN-KF (R values = 0.79, 0.68, 0.75, and 0.49), and were higher than regression (R values = 0.13, 0.48, 0.39, and 0.37) models for shoots and roots from leaf and node explants, respectively. Because ANN models have the ability to interpolate from unseen data, they could be used as an effective tool in accurately predicting the in vitro growth kinetics of Bacopa cultures.

Coupling auxin‐inducible degradation with quantitative proteomics for in‐vivoprotein functional characterization

Functional characterization of proteins typically involves disrupting expression by genetic deletions, mRNA knockdowns, or by inhibiting function with conditional mutations, followed by observation of concomitant biological effects. These methods, however, are prone to indirect effects resulting from the permanent or long‐term changes to the target protein expression/function. In contrast, the auxin‐inducible degradation (AID) system allows very rapid depletion of endogenous target protein levels, thereby minimizing deleterious effects from long‐term perturbation caused by some traditional methodologies. Combining the AID system with quantitative proteomics in applicable model systems allows detection of proteome changes immediately following target protein degradation. The rapid depletion achieved by AID is well‐suited to studying the biological specificity of regulatory enzymes and confidently identifying their direct substrates. Our lab is interested in understanding the role of phosphatases in the regulation of the cell cycle. We demonstrate the power of combining AID and proteomics in the budding yeast, S. cerevisiae, by targeting components of protein phosphatase 2A (PP2A), an abundant heterotrimeric phosphatase with numerous substrates involved in multiple cellular processes, including the cell cycle. The AID system achieves ≥ 85% reduction in the abundance of PP2A components within 10‐15 minutes after auxin addition, resulting in detectable phosphoproteome changes by 20 minutes. The rapid response time allows us to selectively probe the proximate phosphoproteome changes enriched with direct PP2A substrates while minimizing long‐term, indirect effects resulting from slower transcriptional changes and cellular adaptation responses. Current work is focused on applying this system to studying the contributions of different PP2A complexes at different cell cycle stages, providing novel information on PP2A regulatory contributions to this fundamental biological process. The AID system coupled with proteomics is broadly applicable for studying the role of almost any protein in diverse physiological settings and model organisms.

Wound-induced signals regulate root organogenesis in Arabidopsis explants

BackgroundReactive oxygen species (ROS) and calcium ions (Ca2+) are representative signals of plant wound responses. Wounding triggers cell fate transition in detached plant tissues and induces de novo root organogenesis. While the hormonal regulation of root organogenesis has been widely studied, the role of early wound signals including ROS and Ca2+ remains largely unknown.ResultsWe identified that ROS and Ca2+ are required for de novo root organogenesis, but have different functions in Arabidopsis explants. The inhibition of the ROS and Ca2+ signals delayed root development in detached leaves. Examination of the auxin signaling pathways indicated that ROS and Ca2+ did not affect auxin biosynthesis and transport in explants. Additionally, the expression of key genes related to auxin signals during root organogenesis was not significantly affected by the inhibition of ROS and Ca2+ signals. The addition of auxin partially restored the suppression of root development by the ROS inhibitor; however, auxin supplementation did not affect root organogenesis in Ca2+-depleted explants.ConclusionsOur results indicate that, while both ROS and Ca2+ are key molecules, at least in part of the auxin signals acts downstream of ROS signaling, and Ca2+ acts downstream of auxin during de novo root organogenesis in leaf explants.

In Vitro Propagation of Tribulus terrestris with IAA and BAP Concentrations

<p><em>Tribulus terrestris</em> is conventionally propagated using seeds but has limited germination capacity. One approach that can be done for this plant multiplication is in vitro tissue culture. This study was conducted to determine the growth potential of <em>T. terrestris</em> in vitro by the addition of auxin (IAA) and cytokinin (BAP) as growth regulators at several concentrations. The study was conducted at the Laboratory of Plant Physiology and Biotechnology, Faculty of Agriculture, Universitas Sebelas Maret Surakarta from February to December 2017. The experiment employed factorial Completely Randomized Design (CRD) method with 2 factors, namely IAA concentration (0 ppm, 0.1 ppm, 0.2 ppm, 0.3 ppm), and BAP concentration (0 ppm, 0.3 ppm, 0.5 ppm, 0.7 ppm). The observed growth parameters were shoot emergence time, leaf emergence time, root emergence time, number of leaves, and number of roots. Data were analyzed using analysis of variance followed by DMRT with a 95% confidence level. The results showed that the addition of IAA only affects leaf emergence time, while the addition of BAP had a significant effect on the leaf emergence time and number of leaves. The interactions between IAA and BAP had a significant effect on leaf emergence time.</p><p> </p><p align="left">Keywords: explant; plant growth; tissue culture.</p>

Optimization of stages of clonal micropropagation of garden strawberry varieties Nelly and Kemiya breeding of NCFSCHVW

The production of strawberry planting material using the method of clonal micropropagation of plants is promising, modern and already widely used. This paper presents the results of assessing the breeding potential of garden strawberry varieties Nelly and Kemiya (selection of FSBSI NCFSCHVW) in in vitro culture. Apexes from growing rosettes of strawberries served as the starting material for the initiation. The best survival rate of explants was noted during the initiation in the second decade of August, which were in Nelly variety - 90%, Kemiya - 83.3%. At the stage of proliferation, 6-Benzylaminopurine (6-BAP) was injected into the culture medium at various concentrations (0; 0.5; 1.0 and 1.5 mg / L). To increase the efficiency of micropropagation of strawberries Nelly and Kemiya varieties, it is advisable to add 6-BAP into the culture medium with the amount of 1.0 mg / l. In this variant, 10.6 shoots were formed in the explants in Nelly variety, 5.9 in Kemiya variety. With an increase of the concentration of 6-BAP to 1.5 mg / l, the number of vitrified shoots were raised. According to the results of three subcultures, it was noted that the Nelly variety has a higher reproduction potential compared to the Kemiya variety. At the stage of rhizogenesis, the addition of auxins to the medium was not required, since the roots of regenerated plants formed independently.

An in vitro grafting method to quantify mechanical forces of adhering tissues.

Grafting is an indispensable agricultural technology for propagating useful tree varieties and obtaining beneficial traits of two varieties/species-as stock and scion-at the same time. Recent studies of molecular events during grafting have revealed dynamic physiological and transcriptomic changes. Strategies focused on specific grafting steps are needed to further associate each physiological and molecular event with those steps. In this study, we developed a method to investigate the tissue adhesion event, an early grafting step, by improving an artificial in vitro grafting system in which two pieces of 1.5-mm thick Nicotiana benthamiana cut stem sections were combined and cultured on medium. We prepared a silicone sheet containing five special cutouts for adhesion of cut stem slices. We quantitatively measured the adhesive force at these grafting interfaces using a force gauge and found that graft adhesion started 2 days after grafting, with the adhesive force gradually increasing over time. After confirming the positive effect of auxin on grafting by this method, we tested the effect of cellulase treatment and observed significant enhancement of graft tissue adhesion. Compared with the addition of auxin or cellulase individually, the adhesive force was stronger when both auxin and cellulase were added simultaneously. The in vitro grafting method developed in this study is thus useful for examining the process of graft adhesion.

Construction of Tight Conditional Mutants Using the Improved Auxin-Inducible Degron (iAID) Method in the Budding Yeast Saccharomyces cerevisiae.

Conditional mutants, such as temperature-sensitive (ts) mutants, are effective tools for the analysis of essential genes. However, such mutants are frequently leaky. To overcome this problem, it is helpful to isolate a "tight" conditional mutant of a gene of interest, e.g., by using ubiquitin-mediated protein degradation to eliminate the gene product. One such strategy is the auxin-inducible degron (AID) system, which is easy to use because the simple addition of auxin can induce the degradation of a target protein. Sometimes, however, elimination of the target protein is not sufficient, and an AID mutant exhibits a "leaky" phenotype. To address this issue, the improved AID (iAID) system was developed. In this approach, transcriptional repression by the "Tet-OFF" promoter is combined with proteolytic elimination of the target protein by the AID system, yielding a much tighter mutant. Because simple addition of tetracycline is sufficient to repress the Tet-OFF promoter, the combination of Tet-OFF and AID maintains the ease of use of the original AID system. In this manuscript, we describe how to construct and use iAID mutants in the budding yeast Saccharomyces cerevisiae.

Введение в культуру in vitro эндемика Урала Gypsophila uralensis Less. (Caryophyllaceae)

The culture of loose light green callus tissue from seedlings Gypsophila uralensis Less. was obtained using Murashige-Skoog (MS) and Woody Plant Medium (WPM) with the addition of BA 1.0 mg / l + IAA 0.1 mg / l (where BA = 6-benzylaminopurine and IAA = Indolyl-3-acetic acid). Callus had a high morphogenic activity for four passages. The percentage of viable callus varied from 82 % to 94 %. The average number of fragments per callus was 6.2 ± 0.4 on MS and 4.5 ± 0.4 on WPM. With prolonged cultivation (more than eight weeks), the callus darkened and died. The transition from callus proliferation to organogenesis was noted when the MS medium changed to Silene cretacea Saratov (SCS) medium. Further induction of morphogenesis took place on SCS medium with a complex set of growth regulators (BAP 0.2 mg / l + IAA 0.5 mg / l + CIN 1.0 mg / l + HA 1.0 mg / l). Up to 90 % of the callus switched to the formation of adventitious shoots after four to six weeks of cultivation. 2 to 13 shoots developed with a height of up to 2 cm with 2-4 pairs of well-developed green leaves depending on the size of the callus. Rhizogenesis was observed only on the WPM nutrient medium with the addition of IAA auxins from 0.1 mg / l to 1.0 mg / l and IAA 0.2 mg / l + IBA 0.5 mg / l (where IBA = indolylbutyric acid). The beginning of the formation of roots was observed after three to four weeks. The proportion of rhizogenesis was 51–53 %, i. e. both an increase in the concentration of IAA and the addition of IMA to the medium didn`t lead to an increase in the number of microturns with roots. The possibility of obtaining regenerated plants in the callus culture of Gypsophila uralensis was shown.

Study of Ms Media With Addition Of Auxins And Cytokinin on Growth and Development of Arr (Arenga Pinnata (Wurmb) Merr.) Culture.

Aren has an important role as a biofuel producer, so research is needed for the development of this plant. This research has been carried out in the Tissue Culture Laboratory of the Department of Agrotechnology, Faculty of Agriculture, Muhammadiyah University, North Sumatra, from October 2017 to September 2018. This study aims to obtain a combination of concentrations of growth regulators of curcumin and cytokines which are best for increasing plant population in vitro culture. , as well as to produce large quantities of quality plantlets in a relatively short time. Experiments in the form of a Completely Randomized Design (CRD) were repeated 3 times with 5 samples of each treatment to obtain 105 units of experimentation. The first series experiment was the regeneration stage, shoot tip results were cultured on WPM media with a concentration of 0.25 ppm + 1.0 ppm Kinetin which was used as explants. Furthermore, the shoots were regenerated in the WPM medium with the treatment of concentration of growth regulators of auxin and cytokinin which consisted of 7 treatment levels, namely (E0) 0.00 ppm NAA + 0.00 ppm BAP, (El) 0.00 ppm NAA + 0.25 ppm BAP, (E2) 0.50 ppm NAA + 0.00 ppm BAP, (E3) 0.50 ppm NAA + 0.50 ppm BAP, (E4) 0.00 ppm NAA + 0.75 ppm BAP, (E5) 0.50 ppm NAA + 0.75 ppm BAP, (E6) 1.00 ppm NAA + 1.00 ppm BAP. From the observed variables, the percentage of live explants and percentage of explants forming callus the best results were found in a combination of 1.00 ppm NAA + 1.00 ppm BAP (E6) concentration.