Routine lab work, in which 2,4-D is added to the culture medium, generates a considerable amount of waste. For example, one study reported that one laboratory generated about 100 kg of 2,4- D residues in the tissueculture process of plants over a period of three years (SINSKI et al., 2012). However, it is difficult to estimate the amount of residues generated by research institutions by propagating in vitro plants using 2,4-D as auxin. By 2015, 139 biofactories were registered as using planttissueculture techniques for plant propagation in Brazil (CARVALHO et al., 2016). The amount of 2,4-D released in procedures of this type is indeterminant. Nevertheless, from the values reported by Sinski et al. (2012), it can be estimated that the values released into the sewer or trash can be extremely high, considering the activities of laboratories around the world.
Nowadays, there is an increasing interest in the use of medicinal plant, but insufficient information, quality and control of some phytotherapeutical products offered is evident, and may not bring the results expected by professionals and users (VICTÓRIO; LAGE, 2008). Tissueculture techniques enable high efficiency and quick production of ethic and pathogens-free plants; besides provoke a considerable interest as a potential alternative to produce bioactive compounds. Plant growth regulators are fundamental to integrate and regulate plant development and their addition in culture media have been widely employed to improve morphogenesis and plant development (NAMDEO, 2007).
ABSTRACT - The high demand for plant material from Calendula officinalis in the production of herbal medicines and cosmetics, turns the technique of plant-tissueculture into one of the alternatives for the improvement of crops over a short period of time. A protocol for tissueculture was developed from segments of seedlings of C. officinalis, in order to improve the proliferation of shoots and roots. We used a Murashige and Skoog (MS½N) medium, reduced to half the concentrations of NH4NO3 and KNO3 to verify the effect of different types of explants (basal, intermediate, and apical), a medium containing beach sand as support instead of agar, and the effect of auxins and cytokinins (TDZ tidiazuron; BAP, 6-benzylaminopurine, IAA, indol-3-acetic acid, IBA, indol-3-butyric acid, NAA, naphthalene-acetic acid) on plant development in vitro. The results showed pronounced rooting from the apical explants, as well as a greater elongation of shoots and number of leaves. The solid medium was more suitable for the C. officinalis cultures. Shoot proliferation was dependent on cytokinins with better results from the influence of TDZ or BAP compared to the other treatments. Plants regenerated from the medium containing TDZ displayed a glazed appearance and morphogenetic deformations. The highest rate for rooting (80%) was obtained using IAA 0.1mgL -1 . Through in-vitro propagation, healthy C. officinalis plants were
Chemical sterilization represents nowadays an interesting alternative for culture medium sterilization, but in most of micropropagation laboratories autoclaving is considered the most security way for culture medium sterilization, main because the long repeatability of results and that avoid any type of microorganism resistance. However, the use of chemical sterilization with chlorine products, as chlorine dioxide, also represents an important tool to be use in planttissueculture labs for production of thousands of plantlets, and represents one way to reduction costs of micropropagated plantlets, one of the challenges of micropropagation techniques for this century (CARDOSO et al. 2018). Studies with chemical sterilization of also liquid culture media for temporary immersion bioreactor systems needs be realized to evaluate the use and efficiency of chlorine dioxide also for this system, actually used for large-scale micropropagation of plantlets by tissueculture.
Stevia is an important sweetener herb. In normal propagation it is very difficult to regenerate due to various reasons like heterozygous nature, self incompatibility of flowers, lack of efficient fertilization and most importantly poor seed viability, hence there is urgent need to develop efficient protocol for rapid multiplication and thus conservation, because till date no appropriate and efficient method is available for its regeneration. Since tissueculture technology is the only process for the mass propagation, this rapid and efficient regeneration protocol provides a good platform for the multiplication and effective conservation of this important plant.
According to the mRNA levels, seven types of genes (GS, GDH, AlaAT, AspAT, ASN, NADH-GOGAT, Fd- GOGAT) in the DvGS1-1 line showed significantly in- creased transcriptional levels compared with the wild type under either normal or low nitrate conditions. Consequently, the activities of these enzymes (except ASN, as there is no reference describing an approach for ASN assay) together with the NR and NiR, were assayed to test the concordance between mRNA level and enzymatic activity. In leaf tissue, it was observed that the activities of GS, GDH, Fd-GOGAT, AspAT, and AlaAT in the DvGS1-1 line were generally in- creased by 13-21% under both nitrate supplies (Fig. 4A-E, G, I-L). The NR and NiR activities were re- duced by 10-18% (Fig. 4M-P), and the NADH-GO- GAT activity had no significant change compared with the wild type under both nitrate supplies (Fig. 4F, H). In stem tissue, the activities of GS and GDH were increased by 12-16% under low nitrate supply but showed no significant changes under normal ni- trate condition (Fig. 4A-D). The AspAT activity was increased by 14-20%, the activities of Fd-GOGAT and NADH-GOGAT had no significant changes compared with wild type under both nitrate supplies (Fig. 4E-H, J, L). The AlaAT activity was increased by 13% under normal nitrate conditions (Fig. 4I), the NR activity was reduced by 12% under low nitrate supply (Fig. 4O) and the NiR activity was reduced by 18% under normal nitrate supply (Fig. 4N). In root tissue, the activities of GS and NADH-GOGAT were enhanced by 29% and 57%, respectively, under nor- mal nitrate supply (Fig. 4A, F), and the GDH activity was increased by 13% under low nitrate supply (Fig. 4D). The AlaAT activity was reduced by 13% under
The influence of BAP in the oxidation of the explants is dependent of the species or cultivar, besides the used concentration. In Melaleuca alternifolia, the increase in BAP level favored the oxidation in explants cultivated in the liquid and solid MS or WPM medium. The larger oxidation percentage for all the media was observed in 4.4 µM BAP (Oliveira et al. 2010). On the other hand, in Vitis vinifera cv. Merlot, the BAP levels did not influence the oxidation percentage (Carvalho et al. 2011). But meristems of grapevine cv. 'A Dona' cultured in the presence of 2 to 10 µM BAP presented reduced percentage of oxidation (Passos et al. 1985). At the stage of in vitro multiplication, the capacity of the explants to survive, to develop and proliferate is the consequence of several factors, such as genetic, physiological factors and environmental conditions of culture (Kozai et al. 1997). BAP has a great influence in the shoot proliferation of grapevines, as observed in cv. Merlot (Carvalho et al. 2011), cv. Bordô (Ayub et al. 2010) and grapevine rootstock cv. ‘VR043-43’ (Machado et al. 2007). The consistency of the culture medium also exercises fundamental effect on the morphogenesis and on the shoot growth (Karasawa et al. 2002). Nutrients and water present in the double-phase medium have largest ability to be absorbed, due to a greater amount of nutrients and water available; besides, there are no physical barriers in the culture medium, which increases the explant contact with the nutrients and water whereby it usually increases the multiplication rate.
Methyl salicylate was rapidly detected after 7 min by GC/MS, in an intense peak. The use of BAP may induce hydrocarbons biosynthesis, when compared with plants grown in the control medium. These data agree with those reported for leaf volatiles from A. zerumbet cultured in medium containing cytokinin, where data evidenced a large number of hydrocarbons in leaf volatiles when KIN and BAP were applied (Victório et al., 2011). Effects of cytokinins on the level of volatile compounds have been reported in tissue cultures of medicinal plants (Affonso et al., 2007). However, in this study, the addition of cytokinin did not improve methyl salicylate production in vitro cultures of P. paniculata, conversely a reduction in its content was observed in growing concentrations of BAP (Figure 3).
Abstract: Problem statement: The in vitro propagation of plantain (Musa paradisiaca) is still faced with lots of challenges such as blackening or browning of tissues prior to culture due to the oxidation of phenolic compound by polyphenolic oxidase enzyme present in the tissue when excised. Approach: Understanding browning processes in plantain and possible ways of minimizing it during excision of explants with particular emphasis on the use of antioxidants was purposed. Tissues were surfaced sterilized with 0.1% (w/v) HgCL 2 for 1-6 min to get a pure culture and then treated for 2 h with
UHPLC with high resolution Time of Flight mass spec- trometry proved to be a powerful technique for the analysis of the macrocyclic trichothecenes in plant extracts detec- ting 51 different major macrocyclic trichothecenes. The major indings were Baccharin B2/B1/4, Baccharinoid B12/B17, iso-baccharin/baccharin and the conjugate xylo- se-roridin L-2. Out of 14 available trichothecene standards only 2 (roridins E and A) could be detected in the samples. Trichothecenes found in the sample with no possibility of standard matching were identiied tentatively on the basis of the MS/MS with spectra showing a fragmentation pat- tern (accurate mass) consistent with macrocyclic tricho- thecenes (Nielsen et al. 2011). These identiication points included several water loss ions as well as the m/z 231 and 249 ions seen from macrocyclic trichothecenes, or m/z 229 and 247 seen in case of hydroxylation of the trichothecene skeleton. Interestingly neither verrucarins nor roridin L-2 were detected. Expressed as roridin A equivalents the 51 macrocyclic trichothecenes summed up to a total content of 1.1-1.2mg/g which is in the same range found by Jarvis et al (1996) where 0.04-0.7mg/g was detected. Using MS/ HRMS and in-source fragmentation, 15-20% of the total trichothecenes contents was found to be conjugated to a glucose (seen by the loses of a glucose moiety) also found by Jarvis et al. (1996) and one third constituted by 6 aldo- pentose conjugates, probably xylose conjugates, which was also partly conirmed by better retention of these compared to their glucose analogues. These aldopentose derivatives have to our knowledge has never been reported in the li- terature. All these monosaccharide derivatives could easily be identiied as they only produced [M+NH 4 ] + and [M+Na] +
Each of the plant parts (seeds, stem and leaves) as well as the various tissue samples (2,4,6 and 8 weeks old)were dried, powdered, weighed and then separately subjected to soxhlet extraction in petroleum ether for 24 hr on a water bath for removing fatty acids. Each of the mixture was filtered and residual tissue masses were
The extracts were prepared from in natura specimens after the leaves, stems and inflorescences separation. The extracts obtained from the callus were obtained from the entire plant material with no distinction between organs and seedling of P. ornatus. Then, each of these materials were washed and dried in oven with aeration at 40 °C, and subsequently ground. The ground plant material (5.0 g) was immersed in hot water (80 °C) infusion under stirring for 30 minutes and then filtered. The infusion process was repeated once more with the remaining solid residue from filtration. The two filtrates were combined and added HCl 25% (v/v) until the mixture reached pH 2-3 in order to maintain the compounds of interest in acidic form. Thereafter, the mixture was partitioned three times between ethyl ether and H 2 O. The ether portions were dried and
To test the efficacy of the modified solvent system, we also purified Cer (as a single fraction) from the lipid extracts of mouse brain and HOG cells (initially used to determine the precise solvent compositions and to test the efficacy of Cer purification using cell culture). The lipid extract was applied on a silicic acid column (0.7 g) in 1 ml of chloroform:acetone:acetic acid (24:1:0.01; v/v/v, solvent 1), the column was washed with 25 ml of the same solvent, and collected as fraction 1 or F1. Ceramide was eluted as a pure fraction using chloroform:acetone: acetic acid 18:2:0.01 (v/v/v; 15 ml, solvent 2, fraction 2 or F2). The two fractions (F1 and F2) collected separately were analyzed by TLC for their lipid composition. Sphingolipidomics (HPLC-MS) anal- ysis was performed at the lipidomics core facility (directorship of Dr. Jacek Bielawsiki) of the Medical University of South Carolina (MUSC), Charleston, SC.
After 18 days of culture, formation of acidic compartments of osteoclast-like cells was detected by incubation with DAMP followed by immunostaining and CLSM imaging. Fig. 4 shows cell nuclei (blue), actin skeletons (red) and acidic compartments (green) of osteoclast-like cells as well as the blue autofluorescence of the chitosan fibres. The overlap of red and green signals appears as yellow areas, which confirms acid compartments developed within all cells. Green areas without cells refer to acidic enrichments left behind by mobile osteoclast-like cells. Detection of acidic compartments on coated scaffolds is slightly disturbed by interference of red fluorescence of cell actin and collagen. Differentiation of monocytes towards the osteoclastic lineage was additionally analyzed by microscopic detection of the typical markers cathepsin K and TRAP. Fig. 5a shows the cell nuclei (blue), actin skeletons (red) and cathepsin K (green) of osteoclast-like cells after 18 days of cultivation on the uncoated chitosan scaffold. The chitosan fibres occur as light blue in the image due to the autofluorescence. The green fluorescence confirms that cathepsin K is concentrated inside the cells. That is visible more clearly by shielding the red signal of the actin staining (inset of Fig. 5a). TRAP activity of osteoclast-like cells at the same time point is visualized as yellow dots in Fig. 5b. The actin skeleton is stained green. Detection of cathepsin K and TRAP on collagen-coated scaffolds is disturbed by autofluorescence of the scaffold.
of 400 mg of collected joint cartilage. Each hydrogel mea- sured 3 mm in diameter. (Figure 2) After alginate hydrogel po- lymerization, we could see under microscope a homogenous distribution of the chondrocytes in each hydrogel. (Figure 1C). The chondrocytes maintained a spherical morphology similar to the original cartilage (Figures 1C and 1D) when compared to monolayer culture (Figures 1A and 1B), remaining fixed at the polymer. Since the first days of culture, the presence of cell agglomerates on alginate pearls had demonstrated strong cell division, similarly to isogen groups of the newly-formed hyaline cartilage. (Figure 1D) By weekly accessing cell viability on al- ginate pearls stained with trypan blue, the increased cellularity and viability maintenance can be confirmed by comparing it to the number of baseline cells (Figure 3). On the fourth cul- ture week, high cellularity was observed by microscopy, com- pared to the first week, and all hydrogels remained intact, in addition to the maintenance of a rounded phenotype of the chondrocytes.
cells differentiated into myoblasts. As shown in Fig. 5A, myogenic progenitor cells in the presence or absence of serum were randomly oriented at day 4. However, when they underwent terminal myogenic differentiation (at day 14), cells were highly oriented and aligned. Similarly, we have calculated various shape indexes including circularity, aspect ratio, and fusion indices. Figure S1 shows morphological changes of cell nuclei with culture time. At early stages of myogenic differentiation, cell nuclei were found to be more spherical, which is represented as circularity, and had lower aspect ratio (Fig. S1). However, upon terminal myogenic differentiation, the cell nuclei became more elongated and had higher aspect ratios for both serum-containing and serum-free media (Figs. 5B,C). In addition to the nuclei shape, we also calculated the differentiation indices to assess differentiation efficiency and the fusion indices for fully differentiated multinucleated myotubes, demonstrating that there were more differentiated cells in cultures containing serum compared to serum-free medium (Fig. 5D, E).
A trait × environment biplot based on mean values in each of the years was used to visualise similarities/ dissimilarities among tissueculture traits in response to the environment (Figure 1). All tissueculture traits were negatively influenced by environmental conditions in the first year (2003), as evidenced by the obtuse angles be- tween their vectors (the rays connecting the traits to the biplot origin) and the vector of this year. This year was characterized by a higher value of the temperature based parameters than the other two years for the all three pe- riods (Table 2). Rainfall was lower in 2003 compared to 2004 and 2005 for two-week (from flowering to the me- dium milk stage) and six-week (from stem elongation to the medium milk stage) periods prior to sampling of embryos. Hence, warm temperatures and lower water availability in 2003 reduced (p < 0.05) tissueculture per- formance of wheat genotypes compared to more favourable 2004 and 2005 years (Table 2). This is in agree- ment with Hess and Carman (1998) that embryogenic competence of immature wheat embryos is lower when donor plants are exposed to heat or water stress. Inter- estingly, among the 96 genotypes only cultivar Florida (USA) displayed higher (p < 0.05) values of RC and PPE in 2003 compared to 2004 and 2005 (Miti c´ et al., 2009). The authors hypothesised that environmental stresses increased the concentration of ABA in plants of this geno- type, and this induced a better adaptation to the tissueculture and improved the regeneration response. A simi- lar explanation for the observation that donor plants of durum wheat, when subjected to a mild heat or drought
used to eliminate pathogens from infected clonal plant materials and it is based on cryopreservation using vitrification techniques, in which the biological material is exposed to liquid nitrogen (LN) at -196 ºC for a period. Under these conditions, infected vacuolated and differentiated cells are eliminated by the ultra-low temperature effect, leaving only highly cytoplasmic meristematic cells. Cryotherapy has been used successfully in eradicating virus infection from several species with economic importance as plum, banana, strawberry, potato and garlic, besides grapevine (WANG and VALKONEN, 2009a; VIEIRA et al., 2015). High efficiency of cryotherapy in several species, combined with the low operational costs and rapid recovery of plant material without virus infection at a high frequency, have made this method a promising tool for institutions aiming at the recuperation of crops with agronomic potential. Being a new method, there are still few studies in using cryotherapy in comparison with the traditional methods in eliminating viruses, and results on agronomic evaluation of plants that were subjected to cryotherapy are still not available in literature. Despite the benefits and positive results of cryotherapy technique, challenges still remain due to variable response of genotypes within and between species, restricting the applicability of cryotherapy (WANG et al., 2014; FENG et al., 2013). Hence, the next stage of progress in this field will see the development of robust protocols applicable across genotypes, in each studied species, in order to maximize the generation of plants without viral particles. Further evaluation of genetic stability and agronomic behavior of treated plant material is another aspect that will be addressed in cryotherapy research in the coming years.