We had a defined user for the product of our project: small-scale farmers, and we decided that the way we wanted our product to be used is by spraying the AFPs in a solution to the leaves. We bear in mind the legal and social limits of introducing GMOs into the environment in our country, so we always envisioned to release only the protein product. With our proposed implementation comes many challenges regarding the stability of the protein during a whole season of frosts, and about how our product could reach efficiently the interior tissues of the leaves.
We were able to define many aspects of the proposed implementation mainly thanks to Human Practices work and experimental procedures.
The little to medium scale farmers affected by frosts will be the potential end users of our proposed product. It is expected that our product has acceptance, because it is a biological product, GMO free and an innovative proposal made by young peruvian students.
Frost type to target
The crops affected by white frosts will be the type of frosts that could be treated with our product.Know more
Need of protein purification
Experimental procedures indicated that it will be neccessary to purify the AFPs.Know more
Frost type to target
Types of frosts
In order to know deeply about the frost phenomenon in our country, we found that there were two types of frosts commonly known as white and black frosts. The white frosts cause the formation of a layer of ice above plant leaves, meanwhile the black frosts affect the plant internally causing a burn-like aspect. 
An inconvenience is that there is no exact information of how many and which peruvian regions are affected by each type. According to the Assistant Director of Meteorological Prediction from SENAMHI, this data should be taken at each Peruvian region given that our country has a variety of microclimates.
That is why we choose the frost type that affects the farmers we communicate with. They live in a peruvian department called Junin, which suffers mostly from white frosts.
Need of protein purification
The freezing experiments with the culture media showed that the ice-binding proteins (IBPs) of interest, after their expression in P. nigrifaciens, should be purified to apply them directly in the cultures. The reason? They act as nucleating agents. The saline medium for P. bathycetes (PB) and the Luria-Bertani medium (LB) with and without E. coli froze at -5 ° C after shaking (Figure 1). A direct application would make the IBPs have an additional competition with the components of the culture medium. Consequently, the objective of reducing frost damage could be affected. The solution would be to use the purified protein in solution, as it would be free of nucleating agents.
According to literature, AFPs bind to the ice crystals  and given that in white frosts the ice crystals form externally due to the environmental humidity , we consider that spraying would be a proper administration method of our solution.
Consulting the available and/or developing products, there are many patents about mixtures of different compounds for protecting plants from frost and/or freeze and methods of application. Some of these antifreeze agents are sodium chloride, glycerol, glucose, saccharose, urea, ethylene glycol, polyethylene glycol, polyvinylpyrrolidone, block copolymers of polyoxyethylene and polyoxypropylene, and others. However, these types of compounds need to be applied in high dosages .
Spraying is one of the most used methods of application [5-7], but there are variations between patents. One way is applying the formulation as a foliar spray prior to freezing night . Another is spraying via nozzles or sprinkling systems . A third one is to atomize above a field the spray mixtures. This means the formation of droplets due to mechanical action on a liquid medium, by releasing the pressure at small apertures and/or by rotating objects. In addition, 100 to 1000 L of spray mixture are sprayed per hectare. Besides, some equipment for spraying spray mixtures are aircrafts, spraying booms mounted on a tractor, spray lances, and knapsack sprayers .
On the other hand, there are other methods of application such as including the antifreeze agent in normal irrigation water on a long-term basis. Another is to include it in irrigation water, fertilizer formulations, or plant potting soil. Finally, there is the possibility of dispersing the antifreeze agent in the form of a powder on plants .
In that way, we consider that the best administration method is spraying the AFP solution on the plant leaves, reducing the plant injury due to frosts. It fits perfectly with the farmers demand: they want this product to be a liquid solution.
We need to assess the safety of the product before we even present it to the farmers. Thus, first we plan to run lab experiments to test both the independent and joint antifreeze activity of the AFPs. For the future implementation at the Andean region, the selection of the stronger promoter from characterization experiments will allow us to test the AFP expression in the chassis to grow at low temperatures. After the wet lab experiments, we plan to test on greenhouse plants at a gradient of low temperatures. If the results are successful and our solution confers significant protection for the plants, we plan to continue with the field experiments.
On the other hand, we have already contacted agricultural communities affected by frosts and they were predisposed to provide a part of their crops for future experiments, and more importantly their time for an explanation of this developing product. With the Human Practices work, we were able to know their interest in this product, because they need a solution for this recurrent problem.
- SENAMHI. (2010). Atlas de heladas del Perú. FAO - Organización de Las Naciones Unidas Para La Agricultura y La Alimentación, 50.
- Quispe, N. (2020, october). Interview with Assistant Director of Meteorological Prediction from SENAMHI.
- Davies, P. L. (2014). Ice-binding proteins: A remarkable diversity of structures for stopping and starting ice growth. Trends in Biochemical Sciences, Vol. 39, pp. 548–555.
- Agra Group, A.S., assignee. Composition for protecting plants against frost and method of plant protection. European Patent Application EP 2 772 137 A12014. 2014 Sep 03.
- Blum RD, inventor; Egg Factory LLC, assignee. Compositions for protecting plants from frost and/or freeze and methods of application thereof. United States patent US 6,180,562. 2001 Jan 30.
- Fahy G, Wowk B, inventors. Prevention of ice nucleation by polyglycerol. United States patent US 6,616,858. 2003 Sep 9.
- Obata H, Kawahara H, Tomita T, inventors. Control of ice-crystal growth by non-proteinaceous substance. United States patent application US 12/222,863. 2008 Dec 25.
- Blum RD, inventor. Compositions for protecting plants from frost and/or freeze and methods of application thereof. European Patent Specification EP 1 148 781 B1. 2008 Nov 19.
- Klostermann M, Venzmer J, Haensel R, Sieverding E, inventors; Evonik Degussa GmbH, assignee. Use of hydrophobic, self-emulsifying polyglycerol esters as adjuvants and anti-spray-drift agents. United States patent US 10,390,530. 2019 Aug 27