Basic Concepts of Sterilization Techniques

Sterilization, which is any process, physical or chemical, that destroys all forms of life, it is used especially to destroy microorganisms, spores, and viruses. Precisely defined, sterilization is the complete destruction of all microorganisms by a suitable chemical agent or by heat, either wet steam. In this review, we discussed about various suitable techniques that used for removing of infectious agents. The heat sterilization can be applied only to the thermostable products, and chemical sterilization is also used for any types of plastic or glass materials that degrade with heat. The Gas sterilization involves exposing equipment to chemical gases in an enclosed heated or pressurized chamber.

Optimization of explants in vitro sterilization protocol of some deciduous tree species

One of the methods of obtaining planting material of deciduous plants, in particular common ash (Fraxinus excelsior L.), broad-leaved linden (Tilia platyphyllos Scop.) and silver birch (Betula pendula Roth) is microclonal propagation. Asepticity of explants is a prerequisite for microclonal plant propagation. Chemical sterilization with liquid substances is mostly used for this purpose. The mode of decontamination is influenced by a number of factors, in particular the genotype of the plant. The purpose of the study was to optimize the sterilization protocol of F. excelsior, T. platyphyllos and B. pendula explants for microclonal propagation. For research, 20–30 cm of shoots isolated from 12-year-old T. platyphyllos, 10-year-old B. pendula, and 15-year-old F. excelsior in February-March 2021 were used. Plant material was cultured according to conventional methods on a nutrient medium MS (Murashige & Skoog, 1962). Biotechnological methods were used (plant tissue culture in vitro, microclonal propagation). MS Excel software package was used to process the experimental data, the mean and its standard error were calculated. One-way analysis of variance (ANOVA) was performed to analyze the effect of explant sterilization on asepsis. The expediency of keeping plant material during the day in 0.1 % solution of «Samshit»  F. excelsior and 0.3 % solution «Fundazole»  T. platyphyllos and B. pendula is shown. The sterilization protocol of experimental plants (efficiency over 50 %) was optimized by using a stepwise method using 70 % ethyl alcohol, 1.0 % and 2.0 % AgNO3 and 2.5 % and 5.0 % NaClO. The effect of the sterilization regime of experimental plants on asepsis is statistically significant at the level of 5%. To initiate the explants, a culture medium according to the MS prescription was used with the addition of 0.25 mg/L kinetin and 2.0 g/L activated carbon. Further studies are aimed at developing a protocol for direct regeneration of microshoots of F. excelsior, T. platyphyllos and B. pendula under the action of components of the culture medium in vitro.

Acacia Mangium × A. Auriculiformis Micropropagation in a Non-Sterile Environment

Background: Autoclaving is used to eliminate contamination during tissue culturing, however, it is a complicated process, time-consuming and costly. Chemical sterilization of tissue culture can effectively eliminate contamination, is a simple procedure, and cost effective. However, studies on the chemical sterilization mostly focus on bud induction, while the effects of chemical sterilization overall process of tissue culture, including bud induction, proliferation, and rooting, remain to be determined. Here, we investigate the effect of chemical sterilization on bud induction, proliferation, and rooting of Acacia mangium × A. auriculiformis.Results: The results showed that chlorothalonil (0.2 g/L) was a suitable chemical sterilant, and bud induction medium was 1/8 Murashige and Skoog medium + agar 7 g/L + chlorothalonil 0.2 g/L + 6-benzylaminopurine 0.5 mg/L The highest induction rate (99.54%) was observed in the third to fifth buds’ stem segments collected in October treated with 0.8 g/L carbendazim for 3 min, with a contamination rate of 0. The rooting medium was agar 7 g/L + chlorothalonil 0.2 g/L+ indolebutyric acid 1.5 mg/L + naphthylacetic acid 0.5 mg/L, and the rooting rate was 97.62%. The proliferation rate and subculture duration showed a positive correlation, while the proliferation rate was 3.58 times higher at the fourth subculture rooting. Conclusions: Our results suggest that chlorothalonil can effectively replace autoclaving during bud induction, proliferation, and rooting of A. mangium × A. auriculiformis. The findings of this study provide technical support for rapid seedlings propagation, accelerates the breeding process of Acacia, and can be applied in other tree species.

Costreductionin the micropropagation of Solanum lycopersicumL. var.cerasiforme

The aimof this study was to establish a low-cost alternative protocol for micropropagation ofSolanum lycopersicumL. var.cerasiforme,popularly known as cherry tomato. In the in vitroestablishment, culture mediacontaining Laboratory Reagent-grade (LR)and commercialsucrose and varied concentrations of corn starch and agarwere tested. The replacement of thermal sterilization, usingautoclave,withchemicalsterilization,adding sodium hypochlorite (2%) in the medium, was also evaluated. In the multiplication stage,the mediumwas supplemented with agar and/or corn starch and commercial sucrose.Forrooting, agrowth regulator-free medium withcommercialsucrose supplemented with agar and/or starch was used. Themicroplants were thentransplanted into plasticcontainerscontainingonlygarden substrateandsubsequentlyacclimatized in a greenhouse.The results make it possibleto conclude that the reduction of costs in the micropropagation of cherry tomatocan beobtained by replacing LRsucrose with commercial sucrose,and by the use of chemical sterilization of the culture medium withsodium hypochlorite. The replacement of agar withcorn starch can be done partially, in the stages of establishment and multiplication,and totally, during rooting

In Vitro Propagation of the Amazonian Medicinal Plant Guayusa (Ilex Guayusa) and Effects of Light in the Growth and Development of this Shade Tolerant Plant

Guayusa ( Ilex guayusa ) is an endemic plant from the Amazon with potential medicinal applications. Indigenous people are familiar with such applications and use guayusa based on ancestral knowledge. There is a growing interest in guayusa-based products in urban areas of Ecuador and internationally. The supply cannot meet the demand. Currently, traditional practices are used for guayusa growth and the potential use of the protected forest is foreseen. This work describes a protocol for the in vitro propagation of guayusa, a sustainable solution to generate high quality plants in reduced space. Stakes obtained from stems were used as explants. Chemical sterilization with ethanol and sodium hypochlorite resulted in 100% surface-sterilized stakes. The growth medium mWPM resulted in favorable outcomes regarding shoot development and elongation, as well as rooting. Supplementation with activated charcoal resulted in reduced browning during the elongation phase. The majority of shoots were able to develop roots spontaneously. Medium supplementation with the auxin indole-3-butyric acid, IBA, may be considered when rooting does not occur spontaneously. Acclimatization was performed in soil. The protocol was tested under different light spectra, revealing that guayusa growth is affected by light quality. The photobiology of this shade tolerant plant requires further characterization, but the data uncovered a potential role for green and far-red light in root development.

IN VITRO SEED GERMINATION AND PLANT GROWTH OF “CABEÇA-DE-FRADE” (CACTACEAE)

ABSTRACT The genus Melocactus (“cabeça-de-frade”) comprises 32 species in Brazil, of which M. glaucescens and M. paucispinus are threatened with extinction. The present work evaluated the effects of different concentrations of Murashige & Skoog (MS, MS/2 and MS/4) culture medium and sucrose (15 g L-1 and 30 g L-1) on in vitro seed germination and plant growth of M. glaucescens and the efficiency of sterilization with sodium hypochlorite (NaOCl) on in vitro seed germination and plant growth of M. glaucescens when using seeds and M. glaucescens and M. paucispinus when using apical segment of cladode. In M. glaucescens, the final germination at the different MS and sucrose concentrations varied between 53.5 and 68.1% and the best results for in vitro growth were observed with the lowest mineral salt (MS/2 and MS/4) and sucrose (15 g L-1) concentrations, with lengths of the aerial portion of 9.70 and 10.76 mm, respectively. There was no difference in seed germination and plant growth in chemical and autoclave medium. It is concluded that the use of chemical sterilization with NaOCl at low concentrations of salts (MS/2 and MS/4) and sucrose (15 g L-1) are quite advantageous for producing ornamental plants germinated in vitro and/or apical segment of cladode of M. glaucescens andM. paucispinus, representing a reduction of costs for in vitro cultivation of this species.

Transcutaneous Ultrasound Guided Intraovarian Injection in Rats (Rattus norvegicus)

The goal of this study was to develop a method for ultrasound-guided percutaneous intraovarian injection in Wistar rats.Intraovarian administration of chemicals or needle aspiration of the ovary has been undertaken in some species, includinghumans, equines, and bovines. In rodents, which are widely used in scientific research, a technique for intraovarian injectionwithout surgical exposure of the organ has not been described. The current study standardized the procedure of ovarianpercutaneous injection of 0.9% sodium chloride guided by ultrasound in rats. The ovaries were measured by ultrasoundimaging before and immediately after injection and showed a significant increase in ovarian length but not width. No clinical abnormalities were detected within 15 d after injection. These findings indicate that the steps of ultrasound localization of the organ, digital restraint, and correct needle insertion achieved successful intraovarian administration of saline without invasive surgery. These results document the feasibility of ultrasound-guided intraovarian percutaneous injection in rats and may be useful for future research on female reproduction and chemical sterilization.