Project proposal

Tittle: development of transgenic tomato plants for enhanced insulin production

Prepared by: Kiprotich Josphat Siele

Institution: Kabianga University

Email: jsiele183@gmail.com

Phone: 0720436506
Abstract

The development of transgenic tomato plant for enhanced insulin production represents annual

approach to address the rising global demand for insulin, particularly for the treatment of

diabetes.

This study focuses on the genetic engineering of solanum lycopersicum to express a synthetic

gene for proinsulin the precursor of insulin, enabling the plants to produce the crucial protein.

Utilizing agrobacterium-mediated transformation, iw introduce a construct containing the

proinsulin gene under the control of a tissue-specific promoter.

Molecular analysis confirmed the integration and expression of the transgene in multiple tomato

lines, which were then assessed for proinsulin yield through ELISA and Wetsren Blolthig

techniques preliminary results demonstrated significant levels of proinsulin accumulation in

transgenic lines compared to non-transgenic controls. Additionally, optimization of growth

conditions and biotic stress management strategies enhanced overall productivity.

The functional activity of the synthesized proinsulin was evaluated through in vitro active insulin

production, pausing the way for further studies on large –scale cultivation and application.

Ultimately this work contributes to the fields of agricultural bio-technology and metabolic

engineering offering a promising solution for metabolic engineering offering a promising

solution for diabetes management worldwide.

Diabetes mellitus is a chronic metabolic disorder characterized by high blood glucose levels due

to insufficient insulin production or impaired insulin action with the increase prevalence of

diabetes globally; there is significant demand for insulin, a hormone critical for regulating blood

sugar levels. Traditional sources of insulin derived from animal pancreases or recombinant

production (rDNA) in microbial system have limitation including high production costs,

potential for immunogenic reaction and ethical concerns associated with animal derived

products.

In recent years, plant biotechnology has emerged as a promising alternative for the production of

therapeutic protein, including insulin. Plants can offer several advantages over traditional

production systems, such as lower costs, scalability and the ability to perform post translational

modifications similar to those in higher eukaryotes. Among various plant species, tomato

(solanum lycopersicum) has gained attention due its favorable growth characteristics, established

genetic transformation protocols and the potential for high protein yield in fruit.

The strategy for engineering tomatoes to produce insulin involves the introduction of a transgene

encoding proinsuline the precursor form of insulin, the proinsulin molecule undergoes

enzymatic cleavage in the body to produce active insulin making it essential for therapeutic

application. The synthesis can be achieved through agrobacterium mediated transformation

which allows for precise integration of the desired gene into the plant genome. In addition to

enhancing insulin production, this biotechnology approach aligns with the growing interest in

sustainable agricultural and the need to develop crop plants with novel fruits by leveraging the

natural abilities of plants for proteins synthesis and accumulation, researchers aim to create a
reliable and efficient platform for insulin production that could ultimately reduce the costs of

diabetes treatment and improve patient accessibility.

This background underscores the potential impact of developing transgenic tomato plants for

insulin production, representing a convergence of agriculture, biotechnology and healthcare that

could significantly benefit global health particularly in communities heavily affected by diabetes.

LITERATURE REVIEW

Developing transgenic tomato plants for enhanced insulin production is a fascinating area of

research that combines plant biotechnology, genetic engineering and potential biopharmaceutical

applications.

Here’s a summary of the relevant scientific literature that outlines key advances and

considerations in this field

Transgenic plant technology- agrobacterium mediated. Transformation – methods for

introducing foreign genes into plants, particularly the use of agrobacterium tumefaciens, which

has been successfully applied in tomato plants (ituangetal - 2019)

CRISPR/cas 9 technology – recent advancement in genome editing techniques ;ike CRISPR cas

9 have opened a new avenue for precise genetic modification to enhance insulin production

(Zhang et al 2020)

Insulin biosynthesis and expression system – plant based expression system –review article

discuss the feasibility of using plants, specifically tomatoes as bio-factories for producing

pharmaceutical proteins including insulin (Reddy et al 2018)

insulin production. Research focuses on optimizing gene constructs that can be inserted into

tomato plants to enhance insulin synthesis studies have explored using the insulin gene fused

with appropriate signal peptides for proper folding and processing within the plant (Doran et al,

2012)

Metabolic engineering, enhancing protein accumulation literature outlines strategies for

enhancing insulin accumulation in tomato fruits through metabolic engineering including using

specific promoters the drive expression in fruit tissues (Mukhopadhyay et al 2021)

Translational modification; studies have investigated modifying post-translational pathways to

ensure correct folding and glycosylation of insulin in plant cells (perec et al 2021)

Regulatory consideration safety and efficiency.- there’s is ongoing research into the safety and

efficiency of transgenic plants as sources of insulin including toxicological assessment and

allergenicity studies(cao et al 2020)

Regulatory frameworks – reviews discuss existing regulatory frameworks for the approval of

transgenic plants and emphasize the need for rigorous testing and compliance with bioethics

(Teixira et al 2021)

Ethical and economic implication – public perception and acceptance : studies explore the

challenges related to public perception of genetically modified organisms(GMOS) and how it

might impact the commercialization of transgenic insulin producing tomatoes (Garcia et al,

2023)
Cost-effectiveness research discusses the potential economic advantages of using transgenic

plants for insulin production compared to traditional methods like recombinant DNA technology

in microorganisms (Sharma et al 2022)

METHODOLOGY

1. Gene selection and cloning

- identify insulin gene sequences and related peptides

- clone these genes into an appropriate plant expression.

2. Transformation of tomato plants

- use agrobacterium

Tunfaciens –mediated transformation to introduce the construct into tomato plant cells

-regenerate whole plants from transformed cells

3. Screening and analysis

- screening for successful transformation using antibiotics resistance or fluorescence markets

-perform molecular analysis to confirm the integration and expression of insulin genes.

4. Proteins extraction and analysis

Extract proteins from transgenic tomatoes

-analyze using SDS-PAGE and western blotting to assess protein expression level
5. Bio assays for functionality

Conducts biological assays using diabetic animal models to evaluate the efficiency of plant

produced insulin.

Objectives

1. Genetic modification – to introduce insulin gene sequences into tomato plants using CRISPR

cas 9 technology agrobacterium – mediated transformation.

2. Expression analysis – to assess the expression levels of insulin proteins in transgenic tomato

plants using techniques such as RT-QPCR and western blotting.

3. Functional characterization – to evaluate the bioactivity of insulin produced in the transgenic

tomatoes plants through in vivo tests on diabetic animal models.

4. Sustainability assessment – to analyze the environment impact and sustainability of producing

insulin through transgenic plants compared to conventional methods.

Justification of the research

1. rational for using tomato plant as a production system

- Climatic adaptability – tomatoes are widely cultivated and can grow in various climates. This

makes them an accessible crop for insulin production in numerous regions.

- High yields- the tomato plant can produce a significant biomass, leading to higher yields of

insulin compared to microbial systems or non-plant eukaryotes.

culturally accepted, which may facilitate the acceptance of transgenic products.

-Ease of genetic manipulation – the tomato genome has been well characterized and genetic

transformation techniques are well established facilitating efficient development of transgenic

variant.

2. Project benefits

- costs-effective production – by utilizing a plant based system, production costs can be

significantly lowered with potential reductions in fermentation and purification costs associate

with traditional methods.

-Scalability – tomato plants can be cultivated on a large scale allowing for the mass production

of insulin with a relatively small environmental footprint.

-Storage and stability – insulin produced in tomatoes may be a more stable stae reducing the

need for refrigeration and enhancing transport options.

3. Health and economic impact

Addressing diabetes with diabetes being a leading cause of morbidity and mortality globally,

enhanced production of insulin can play a critical role in managing this disease improving patient

outcomes and quality of life.

-affordability – reducing the costs of insulin production can lead to lower prices for diabetic

patients making life-saving medications more accessible especially in low-income regions.

agriculture, biotechnology and distribution.

4. Environment consideration

- Sustainable productions – tomato cultivation can be integrated into existing agricultural

systems with potentially less impact on environmental resources compared to industrial scale,

microbial fermentation process.

Reduced wastage – the by-products of tomato cultivation can be utilized in various ways e.g food

for livestock or compost, reducing overall waste from production process.

5. Research and development alignment

Advancement of biotechnology – this project aligns with ongoing efforts in biotechnology. The

enhanced agricultural outputs and develop bio-pharmaceuticals plants.

-Regulatory compliance and innovation-

Development transgenic crops faces stringent regulations hence this project would emphasize

compliance for agricultural biotechnology innovations.

Expected outcomes

- Transgenic tomato plants that successfully express insulin and insulin- like peptides

- Identification of optimal growth conditions for maximal insulin yield.

- Evaluation of the safety and efficiency of plant-derived insulin in pre-clinical models.

- A comprehensive report on the feasibility and scalability of insulin production in

tomatoes.