Hairy roots are a plant-tissue culture raised by Rhizobium rhizogenes infection (formerly known as Agrobacterium rhizogenes). Nowadays, these roots have been gaining more space in biotechnology due to their bene ts for the recombinant expression of valuables proteins; it includes simpli ed downstream processing, protein rhizosecretion, and scalability in bioreactors. However, due to methodological inconsistency among reports, the tissue platform is still a disruptive technology. In the current paper, we propose the rst step to overcome this issue through a systematic review of studies that employ Nicotiana hairy roots for recombinant expression. We conducted a qualitative synthesis of 36 out of 387 publications initially selected. Following the PRISMA procedure, all papers were assessed for exclusion and inclusion criteria. Multiple points of root culture were explored, including transformation methods, root growth curve, external additives, and scale-up with bioreactors to determine which approaches performed best and what is still required to achieve a robust protocol. The information presented here may help researchers who want to work with hairy roots in their laboratories trace a successful path to high recombinant expression.

1. Ant nests are ecologically important emission sources of CO 2 , CH 4 , and N 2 O. An updated review of the progress in studying greenhouse gas (GHG) flux from ant nests could provide a more comprehensive understanding of their role in this context.2. We evaluate the progress in assessing GHG flux assessment through a systematic review and identify factors responsible for higher GHG emissions from ant nests.3. The specific goals were to conduct a bibliographic analysis of the frequency and geographical distribution of scientific works addressing this topic, reported species, and methodologies; to relate the mean GHG flux to species-specific characteristics of ant nests; and to identify patterns and biases responsible for reported higher GHG levels.4. More data is needed on the species and habitats studied. The most frequently examined gas was CO 2 , and the closed chamber system was the most used method, with a wide variation in chamber size and material. No relation was found between CO 2 emissions and species-specific characteristics,which can be explained by the fact that these data show a high variability, probably due to the abiotic factors in each study, different measurement methods, and their respective configurations. 6. Our study underlines the need to standardise GHG measurement methods to allow for reliable comparisons between ant species and habitats. Furthermore, it signals the need to investigate more information to build appropriate global GHG emissions models.7. This progress will only be possible through collaborative studies by increasing interaction among researchers through projects on a continental or global scale.

Greenhouse gas (GHG) emission is a worldwide concern, making the identification of emission sources and their magnitude an urgent issue. Ants release large amounts of carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O) from their nests. Beyond the already known anthropogenic sources of CO2 emissions, such as fossil fuel combustion in the power generation, industrial, residential, and transport sectors, the evaluation of GHG emissions from ant nests is a current research target. However, there are still gaps regarding the real impact of ant nests on GHG emission, primarily because most surveys were conducted excluding anthropogenic areas. In the present study, we aimed to fill part of these gaps by evaluating the magnitude and drivers of GHG emission from ant nests. Our results were organized over three chapters. First, chapter I discussed the current scenario and highlighted the methodological issues of the studies on GHG emissions from ant nests using a systematic review as methodology. We present a scientometry of available publications, which revealed the lack of investigations of ant nests emission in anthropogenic areas and on countries that are higher sources of anthropogenic GHG. Second, in Chapter II, we’ve made a methodology test related to the closed chamber method, guided by some warnings which stood out in the systematic review. Results revealed a dilution effect of the soil surface over the CO2 measurements when using chambers whose diameters are significantly larger than the nest hole. Finally, in Chapter III, we’ve made field measurements of CO2 emissions from Acromyrmex subterraneus nests in urban areas, considering the effect of features of the nest. This investigation revealed an association of CO2 flux with the function of the nest hole, a component of ant nests not yet investigated. Obtained results also showed that ant nests are on a par with recognized anthropogenic CO2 sources, adding one more point to consider in the context of climate change, considering the facility of installation of some ant species in urban and disturbed areas. We hope that these results contribute to expanding knowledge regarding the role of ant nests in CO2 emissions and could even guide the next steps for future investigations.

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Hairy roots are a plant-tissue culture raised by Rhizobium rhizogenes infection (formerly known as Agrobacterium rhizogenes). Nowadays, these roots have been gaining more space in biotechnology due to their benefits for the recombinant expression of valuables proteins; it includes simplified downstream processing, protein rhizosecretion, and scalability in bioreactors. However, due to methodological inconsistency among reports, the tissue platform is still a disruptive technology. In the current paper, we propose the first step to overcome this issue through a systematic review of studies that employ Nicotiana hairy roots for recombinant expression. We conducted a qualitative synthesis of 36 out of 387 publications initially selected. Following the PRISMA procedure, all papers were assessed for exclusion and inclusion criteria. Multiple points of root culture were explored, including transformation methods, root growth curve, external additives, and scale-up with bioreactors to determine which approaches performed best and what is still required to achieve a robust protocol. The information presented here may help researchers who want to work with hairy roots in their laboratories trace a successful path to high recombinant expression.