Despite the advancements in technology and science, Malnutrition is still a global concern. The fact sheet published by WHO suggests that around 45% of deaths among children under 5 years of age are linked to Malnutrition. These primarily occur in low and middle-income countries. The developmental, social, economic, and healthcare impacts of Malnutrition are severe and lasting for individuals, families, communities, and nations . Thus, finding a cost-effective and nutritional source of food has become the need of the hour. Cottonseed is one such nutritious source of food that has the potential to meet the global protein requirements. However, it is not advisable for direct consumption due to the presence of Gossypol.
Our project aims at solving this problem and making cottonseed accessible for direct consumption. In our belief, we have a tremendous social and moral responsibility in solving this problem using synthetic biology.
Our country India is one of the largest producers of cotton globally. Thus, the success of our project would be hugely beneficial to the local communities and farmers whose primary source of income is the cultivation and production of cotton and its products. Our team was highly motivated to create a project that would be environment friendly, beneficial, and accessible to local communities. The prime stakeholders of our project are farmers, researchers, and small-scale cotton processing industries. We set out to interact with people representing these diverse stakeholders.
We interacted with local farmers who cultivate cotton to understand the usage of Cottonseed and Cottonseed meal. We also analyzed the extent of awareness of farmers on Gossypol. We interacted with Prof. Keerti Rathore from Texas A&M University. Prof. Rathore has been working on Ultra-Low Gossypol Cottonseed to make cottonseed safe for consumption. We approached oil processing industries to get insights into cotton oil processing. We conducted surveys to gauge the attitude of people towards nutrition and the extent of awareness and knowledge about the nutritious value of Cottonseed and Gossypol.
Malnutrition is still a significant problem in many parts of the world. Tireless efforts are being carried out to find a food source that can meet the protein requirements of the rapidly evolving population. Many of such food sources are in the form of Genetically Modified Organisms (GMOs). While GMOs are crucial in achieving the global nutrition targets, many people are still skeptical about the safety elements of GMOs.
Our survey aims at collecting data from people on their perceptions of malnutrition, opinions on the introduction of GMOs as a nutritional source of food, and the extent of awareness on Cotton products and Gossypol present in cottonseed.
The majority of respondents belong to Asia. Fortunately, our sample consisted of a large percentage of respondents aged below 25 years, which happens to be our primary target group. A large proportion of respondents have an undergraduate degree as their minimum education qualification. Survey results indicate 33% of female and 67% of male respondents.
Awareness of Cotton and its products
We wanted to collect statistics on the extent of usage of cotton and its products. The majority of the respondents were using generic products derived from cotton, like cotton garments, etc. However, nearly 45% of the respondents were unaware that products derived from cotton, such as cottonseed oil, are edible. About 73% of respondents have never visited a cotton farm or a cotton processing industry.
Awareness of Hunger and Malnutrition
The survey suggested that nearly 97% of respondents agree that hunger is a significant problem to be addressed. 77.3% of the respondents felt that malnutrition is a significant problem in their respective countries.
Opinions on Genetically Modified Organisms (GMOs)
The majority of the respondents had a positive perception of GMOs. More than 86% of the respondents opinionated that GMOs would be applicable to solve the problem of hunger. However, a significant part of the respondents was unsure if GMOs would be used widely as a protein source in the future.
Awareness on Gossypol
Since our project deals with Gossypol, We wanted to understand the extent of awareness about Gossypol among the respondents. More than 77% of the respondents were unaware of the presence of Gossypol in cottonseed. Nearly 59% of respondents did not know Gossypol. Around 30% had little knowledge about the toxin. As a consequence of this, 86% of respondents were unaware of the ill effects of Gossypol.
Opinions on the future of cottonseed
Around 77.3% of the respondents had a positive perception of cottonseed being an efficient and consumable protein source in the future.
Field trip to Cotton farms
Initially, we planned a team visit to cotton farms present around Warangal. Soon after, there was an outbreak of the second wave of COVID-19. Due to the restrictions that subsequently followed, we had to cancel our plans. However, as the situation improved, some of our team members visited cotton farms. Farmers walked us through the cotton cultivation process. We observed various varieties of cotton seeds used for cultivation. We learned about the evolution of cotton plants from seed to fiber. We were inspired by these observations and utilized these observations to improve our project.
Fig: Here are a few images from our trip to a cotton farm.
Captured by our team member Purna.
iGEM is a financially demanding project. Although our institute supported us by providing funds, we still needed some financial support to back our project. Sponsorship was a crucial part of our project due to expenditure on experimentation, lab equipment, materials, etc. Thus, we planned to reach out to companies and individuals for sponsorship. We started a crowdfunding campaign through the platform Ketto. We approached our alumni for fundraising through social media. This fundraising campaign also helped in a better outreach of our project, and as a result, we also received some ground inputs on the project.
Integrated Human Practices
Interaction with Prof. Keerti Rathore
Prof. Keerti S. Rathore Professor, Institute of Plant Genomics and Biotechnology, Texas A&M University.
"Worldwide adoption of Low Gossypol Cottonseed can potentially help meet the protein requirements of over 500 million people"
Nearly 75-95% of calories consumed by Asians come from protein. A familiar food source of Asia, rice has little protein.
In seed and flower petals, the predominant terpenoids present in glands is Gossypol, whereas, in green color tissues, the terpenoids are not just Gossypol but hemi gossypol and hellocide.
Minimal amounts of Gossypol are essential for cotton plants to protect themselves from insects. (However, excessive gossypol content makes cottonseed harmful for consumption).
By experimentation, it was observed, regular cotton contained ~10,000 ppm gossypol. Glandless cotton contained ~10 ppm but had no insect protection. Thus, farmers were not interested in growing it.
Engineered cotton showed ~350 ppm (FDA limit of Gossypol for safe human consumption ~450 ppm) and with insect-protected foliage and floral tissue.
Incorporation into our project
Prof. Rathore was successful in removing Gossypol from the seed. However, complete removal meant that the seed lacked resistance to pests since Gossypol does that work. Thus, his solution is viable in developed countries where storage facilities are good. But in developing countries with minimal storage facilities, this solution is risky. Prof Rathore also emphasized that farmers were not ready to adopt such Gossypol-free cottonseed. These inputs from Prof. Rathore helped us to establish our goal, i.e., to work on reducing the concentration of Gossypol but not its complete removal.
Prof. Rathore also briefly discussed the anatomy of the cottonseed and the sites at which Gossypol is present. This explanation gave us a clear idea about our target sites on the cottonseed.
Prof. Rathore emphasized the importance of a reliable protein source in middle and low-income countries like India. He said that certain enzymes present in the gut of ruminants degrade Gossypol. However, in the case of non-ruminants, Gossypol levels remain a serious problem. He emphasized his research and stated that they used a seed-specific promoter to conduct RNA silencing of delta Cadinene synthase gene. The promoter got active after six days post-anthesis. And Gossypol began to synthesize and hit its peak synthesize rate after 30 days. They made sure that this promoter was not operative in other parts of the plant except embryo seeds.
He also mentioned that his research group tried using silencing technology of antisense, but the results were not satisfactory. Thus they shifted the approach to RNA Interface (RNAi). The Delta Cadinene Synthase (d-CS) enzyme present in the seed is responsible for the conversion of Fameyldiphosphate (FDP) to (+) d-Cadinene. Thus, inhibiting this enzyme is a crucial step towards degrading Gossypol. DNA was isolated from all kinds of cottonseed, and Protein Conversion Ratio (PCR) was obtained to analyze the levels of gene RNAi present in the seed. Some seeds did not have RNAi and showed normal levels of Gossypol. No reduction of Gossypol was observed on the green parts of the plant.
He suggested we explore enzymes that have the potential to degrade Gossypol and narrow the selection down to an efficient enzyme. He gave us some suggestions on the biosynthetic pathways of Gossypol. He also emphasized the importance of Low-Gossypol Cottonseed to meet the nutritional requirements.
Keerti S. Rathore, Devendra Pandeya, LeAnne M. Campbell, Thomas C. Wedegaertner, Lorraine Puckhaber, Robert D. Stipanovic, J. Scott Thenell, Steve Hague & Kater Hake (2020) Ultra-Low Gossypol Cottonseed: Selective Gene Silencing Opens Up a Vast Resource of Plant-Based Protein to Improve Human Nutrition, Critical Reviews in Plant Sciences, 39:1, 1-29, DOI: 10.1080/07352689.2020.1724433
Interactions with Cotton farmers and Cotton processing industries:
It is crucial to share the ideas with the stakeholders and get their inputs to customize the project with a real-world vision. Cotton-producing Farmers are one of the most prominent stakeholders in our project. Thus, few team members reached out to the farmers in and around Telangana, the 3rd largest cotton-producing state. We interacted with them to know more about the cotton cultivation and production process. They told us that the Cotton is a Kharif crop and sown at the same time as rice, in April-May. The typical cotton cycle takes 6-8 months to get the fully developed cotton balls. They also stated that cotton plants are susceptible to adverse environmental conditions, and hence they are harvested before winter frost which could damage the crop and yield. They asserted that the only part of the plant they are interested in is the cotton balls, but it gets damaged on a large scale due to bollworms and other cotton-feeding insects, which is a vital loss to their whole effort. We also created awareness among farmers about Gossypol. So we elaborated our project in the simple layman language and educated them on our project idea. Surprisingly, we got to know that they use the leftovers as feedstock for their cattle.
Because of the restrictions due to COVID-19, we were unable to visit cotton processing Industries. However, we reached out to the manager of a cotton processing industry near Warangal for a telephonic interview to know more about cottonseed oil production. We were briefed about the processes involved in cottonseed oil production. Oil production starts from cleaning the cottonseed to remove impurities. Then seeds are cracked into uniform pieces. The cracked cottonseeds are broken down to uniform smaller flakes with thickness less than 0.5 mm. These flakes are cooked to get rid of excessive water content. The cooking process changes the seed protein to make the oil molecule ready for easy pressing. Thus, obtaining a better quality cottonseed cake and oil. Then the flakes are passed through an oil expelling press machine to extract more oil. The oil thus produced is refined. Impurities and waste materials are separated. In Cottonseed oil production, Cottonseed meal is also obtained as a byproduct after ginning, crushing, and the oil extracted. The cottonseed meal thus obtained is usually used for animal feed and as organic fertilizer. The manager also mentioned that the industry uses solvent extraction and other chemical methods to reduce gossypol levels. He also stated that the chemical method used for gossypol reduction would harm the environment.
Incorporation into our project
The farmers suggested that bollworms and other insects feed on cotton. We wondered if there was a gossypol degradation mechanism in their gut. Thus, we explored this possibility as well referring to the scientific literature. The manager of the industry emphasized the environmental concerns of using chemical methods to degrade gossypol. Thus, we gave a lot of importance to ensuring that our project would be safe for the environment.