modify tomato so that the leaves are edible by humans - Your Published Bio Team Output
**Pre-Project**One potential endogenous pathway that might be useful to modify in this scenario is the biosynthesis of chlorophyll in tomato plants. Chlorophyll is responsible for the green color and bitter taste of plant leaves, so by reducing or altering the production of chlorophyll, the leaves of the tomato plant may become more palatable for human consumption. This modification could be achieved through gene editing techniques to target and modify the genes that control chlorophyll synthesis in tomato plants.
As for a pathway from a different species to import, one potential option could be the edible leaf pathways found in some Asian vegetable varieties, such as bok choy or napa cabbage. These species have naturally evolved to produce edible leaves that are not bitter, and have a more desirable texture and flavor for humans. By importing these pathways into tomato plants, it may be possible to stimulate the production of similarly edible and palatable leaves. For example, specific genes involved in the biosynthesis of key compounds responsible for the mild flavor and texture of Asian vegetable leaves could be identified and introduced into the tomato genome.
Overall, the main modifications to investigate in order to achieve the goal of edible tomato leaves for humans would involve targeted gene editing to modify chlorophyll production, as well as the importation of specific genes or pathways responsible for the desirable flavor and texture found in Asian vegetable leaves. It is important to note, however, that ethical considerations must always be taken into account when modifying the genetic makeup of living organisms, and careful testing and regulation should be employed to ensure the safety and environmental impact of any such modifications.
**Genes:** Genes to add:
1. Arabidopsis thaliana LSL1: This gene is involved in the biosynthesis of lutein, a carotenoid pigment that is found in high levels in edible leaves such as spinach and kale. Introducing this gene to tomato plants could increase lutein production in the leaves, which has been shown to have potential health benefits such as reducing the risk of cataracts and age-related macular degeneration.
2. NAPA1: This gene is involved in the biosynthesis of indole-3-acetic acid (IAA), which is a key plant hormone that regulates growth and development. By introducing this gene, it may be possible to stimulate leaf growth and development in tomato plants, leading to the production of larger and more substantial leaves that are easier to harvest and consume.
3. Arabidopsis thaliana DOF1.1: This gene is involved in regulating carbon and nitrogen metabolism in plants, and has been shown to be important for leaf development and morphology. By adding this gene to tomato plants, it may be possible to stimulate the growth of more succulent and tender leaves that are more palatable for human consumption.
4. Solanum lycopersicum VTE4: This gene is involved in the biosynthesis of vitamin E, which is an important nutrient with antioxidant properties. By introducing this gene to tomato plants, it may be possible to increase the amount of vitamin E in the leaves, providing potential health benefits for humans who consume them.
5. Spinacia oleracea L-galLDH: This gene is involved in the biosynthesis of ascorbic acid (vitamin C), which is an important nutrient with antioxidant properties. By introducing this gene to tomato plants, it may be possible to increase the amount of vitamin C in the leaves, providing potential health benefits for humans who consume them.
Genes to tweak:
1. CHLH: This gene is involved in the biosynthesis of chlorophyll, which is responsible for the green color and bitter taste of plant leaves. By using CRISPR/Cas9 gene editing technology to downregulate the expression of this gene in tomato plants, it may be possible to reduce the amount of chlorophyll in the leaves and make them more palatable for human consumption.
2. NOR: This gene is involved in regulating gene expression in response to light. By using RNA interference (RNAi) technology to downregulate the expression of this gene in tomato plants, it may be possible to stimulate leaf growth and development, leading to the production of more substantial leaves that are easier to harvest and consume.
3. PIF4: This gene is involved in regulating plant growth and development in response to light. By using RNAi technology to downregulate the expression of this gene in tomato plants, it may be possible to stimulate leaf growth and development, leading to the production of more substantial leaves that are easier to harvest and consume.
4. PAP1: This gene is involved in regulating the biosynthesis of anthocyanin pigments that give plants their purple or red coloration. By using CRISPR/Cas9 gene editing technology to upregulate the expression of this gene in tomato plants, it may be possible to increase the production of anthocyanins in the leaves, potentially providing health benefits and making the leaves more attractive for human consumption.
5. GLK1: This gene is involved in regulating the expression of genes involved in photosynthesis, and has been shown to play a role in leaf morphology and development. By using RNAi technology to downregulate the expression of this gene in tomato plants, it
**Regulatory Elements:** may be possible to alter leaf morphology and make them more tender and palatable for human consumption. Additionally, downregulation of this gene may also result in the reduction of chlorophyll in the leaves, further improving their taste and texture.
Promoters:
1. The ubiquitin promoter from Solanum lycopersicum: This promoter has been shown to be highly active in many parts of tomato plants, including leaves. Using this promoter to drive the expression of the added genes, such as LSL1 and NAPA1, may ensure high levels of gene expression in the leaves, leading to increased lutein and IAA production and possibly larger and more succulent leaves.
2. The 35S promoter from Cauliflower mosaic virus: This promoter is commonly used in genetic engineering projects and has been shown to be highly active in many different plant species. Using this promoter to drive the expression of the added genes, such as DOF1.1 and VTE4, may ensure high levels of gene expression in the leaves, leading to increased vitamin E production and possibly more palatable and healthy leaves.
3. The CaMV 35S promoter variant from Solanum chacoense: This promoter variant has been shown to be highly active in tomato plants, including leaves. Using this promoter to drive the expression of the added genes, such as Spinacia oleracea L-galLDH and PAP1, may ensure high levels of gene expression in the leaves, leading to increased vitamin C and anthocyanin production and potentially more appealing and nutritious leaves.
Enhancers:
1. The synthetic enhancer Zm13 from Zea mays: This enhancer has been shown to enhance gene expression in various plant species, including tomato. Using this enhancer in combination with the chosen promoters may further boost gene expression levels in the tomato leaves, leading to desired outcomes such as increased lutein, vitamin E, and vitamin C production.
2. The chimeric enhancer of the CaMV 35S and HSP18.2 promoters from Arabidopsis thaliana: This enhancer has been shown to be active in various plant tissues and can enhance gene expression levels when used in combination with some promoters. Using this enhancer in combination with the chosen promoters may stimulate leaf growth and development, leading to the production of more substantial and palatable leaves.
3. The chloroplast transit peptide enhancer from Solanum lycopersicum: This enhancer has been shown to enhance the expression of genes targeted to the chloroplast, the site of photosynthesis in plant cells. Using this enhancer in conjunction with the chosen promoters may increase the efficiency of the added genes in chloroplasts and result in increased production of nutrients such as lutein and vitamin E in the tomato leaves.
Terminators:
1. The nos terminator from Agrobacterium tumefaciens: This terminator sequence is commonly used in genetic engineering applications and has been shown to be effective in many plant species. Using this terminator to finish the transcription of the added genes in the tomato leaves may ensure that the genes are expressed at the intended levels and do not interfere with other plant processes.
2. The rbcS terminator from Arabidopsis thaliana: This terminator sequence is derived from the gene encoding ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), a key enzyme in photosynthesis. Using this terminator in combination with the chosen promoters may result in more efficient expression of the added genes in the tomato leaves, as it is derived from a gene commonly expressed in leaves.
3. The pea plastocyan
**Vector & Delivery:** in 2A terminator: This terminator sequence is derived from the gene encoding plastocyanin, a key protein involved in electron transfer during photosynthesis. Using this terminator in conjunction with the chosen promoters may enhance the expression of the added genes in chloroplasts and result in increased production of nutrients such as lutein and vitamin E in the tomato leaves. Additionally, this terminator may ensure proper termination of transcription and prevent interference with other plant processes.
Vector:
I would choose the Agrobacterium-mediated transformation method to introduce the desired genetic modifications in tomato plants. Agrobacterium tumefaciens is a soil bacterium that has been shown to transfer DNA to plant cells via a natural process, making it a useful tool in genetic engineering. The advantage of this method is that Agrobacterium can deliver large pieces of DNA to the plant genome, allowing for the efficient transfer of multiple genes at once. The use of Agrobacterium as a vector system also eliminates the need for particle bombardment or electroporation, which can damage plant cells and reduce transformation efficiency.
For this particular project, I would use a binary vector containing the desired genes, regulatory elements, and terminator sequences, along with selectable markers for identifying and selecting transformed plants. The binary vector would also contain the T-DNA borders required for integration into the plant genome via Agrobacterium transfer. The use of a binary vector enhances the stability and versatility of the system, enabling the introduction of multiple genes and the ability to screen for transformants using selectable markers.
Species:
The tomato plant variety ‘Roma’ would be an ideal choice for this project as it is a widely cultivated variety with a compact growth habit and a relatively short lifespan, making it easier to manipulate for transformation and experimentation. ‘Roma’ also has a high yield potential, making it suitable for mass production of edible leaves. Finally, ‘Roma’ is a diploid species, meaning it has two sets of chromosomes, making it easier to manipulate genetically compared to polyploid species.
In conclusion, the use of Agrobacterium-mediated transformation with a binary vector containing the desired genes, regulatory elements, and terminator sequences would optimize the genetic modifications desired for creating edible tomato leaves. The choice of ‘Roma’ tomato plant as the species would provide an ideal growth environment, making it easier to manipulate and mass-produce edible leaves containing lutein, vitamin E, vitamin C, and anthocyanin.
**Selection Marker:** If a selection marker is deemed needed for this project we will use the nptII gene encoding for kanamycin resistance. This marker is commonly used in Agrobacterium-mediated genetic transformation, and it allows for the selection of transformed plants through the use of kanamycin-containing media. The successful transformation and integration of the T-DNA containing the desired genetic modifications into the tomato genome will confer kanamycin resistance, allowing for the selection of transformed plants while avoiding the use of antibiotic resistance markers. The inclusion of a selection marker will facilitate the identification of successfully modified tomato plants, increasing the efficiency of the transformation process and ensuring the desired phenotypic changes are present in the resulting tomato plants.
**Transformation Protocol:** Protocol for transforming tomato plants with desired genetic modifications:
Materials:
- Tomato ‘Roma’ plantlets (preferably in vitro propagated)
- Agrobacterium tumefaciens strain suitable for the vectors being used
- Binary vector containing desired genes, regulatory elements, terminator sequences, and selectable markers
- LB agar plates (with appropriate antibiotics)
- Sterile NaCl solution (0.9%)
- Sterile water
- Murashige and Skoog (MS) medium (with appropriate antibiotics)
- Kanamycin (if using selection marker)
- Sterile filter-tips and pipettes
- Sterilized forceps and scalpel
- Growth chamber/ greenhouse
Procedure:
1. Culture the Agrobacterium strain with the binary vector on an LB agar plate with appropriate antibiotics and incubate overnight at 28°C.
2. Inoculate a single colony from the overnight culture of Agrobacterium in 5 ml of LB medium with appropriate antibiotics and incubate for 16-24 hours at 28°C with constant shaking.
3. Wash the Agrobacterium cells twice in a sterile NaCl solution (0.9%) by centrifuging at 3000 rpm for 10 minutes and resuspending in the same solution.
4. In a new culture tube, mix 200 µl of the Agrobacterium suspension with 2 ml of MS medium containing appropriate antibiotics and 150 µM acetosyringone. Shake gently and let it incubate at room temperature for at least 3 hours.
5. Sterilize the tomato plantlets by washing them in sterile water for 10 minutes, followed by a 15-minute treatment with 70% ethanol and rinsing them 3 times in sterile water.
6. Collect the sterilized tomato plantlets with sterilized forceps and scalpel and cut off the cotyledons and hypocotyls.
7. Transfer the plantlets onto a new MS medium plate containing appropriate antibiotics and wait for 2-3 days for the plantlets to recover.
8. Take 3-4 fully expanded leaves from the plantlets for transformation (avoid using the first two leaves) and throw away the rest of the plantlet.
9. Using a wide-bore pipette tip, puncture the abaxial surface of the tomato leaves to create small wounds without damaging the veins.
10. Dip the wounded tomato leaves into the Agrobacterium suspension, taking care to ensure that the suspension completely covers the wounded area of the tomato leaves.
11. Co-cultivate the tomato leaves with Agrobacterium in the dark at 25°C for 2-3 days.
12. After co-cultivation, place the tomato leaves onto a new MS medium plate containing appropriate antibiotics and incubate for 2-3 weeks in the growth chamber/greenhouse.
13. Transfer the successfully transformed tomato leaves to a new MS medium plate containing kanamycin (if using selection marker) and wait for 2-3 weeks for the formation of new shoots.
14. When new shoots are 1-2cm long, transfer them to fresh MS medium plates containing appropriate antibiotics and wait for the plantlets to grow.
15. Once the plantlets have reached a suitable size, they can be transferred to soil for further growth and experimentation.
By following this protocol, tomato plants can be successfully transformed with the desired genetic modifications using Agrobacterium-mediated transformation. The inclusion of a selection marker (if needed) further facilitates the identification of successfully modified tomato plants, increasing the efficiency of the transformation process and ensuring the desired phenotypic changes are present in
**Gene cassette**: The 5' to 3' list of elements in the multi-purpose cassette are as follows:
1. Ubiquitin promoter from Solanum lycopersicum
2. LSL1 gene from Arabidopsis thaliana
3. NAPA1 gene
4. 35S promoter from Cauliflower mosaic virus
5. DOF1.1 gene from Arabidopsis thaliana
6. Nos terminator from Agrobacterium tumefaciens
7. CHLH gene with downregulated expression using CRISPR/Cas9 gene editing technology
8. Zm13 enhancer from Zea mays
9. NOR gene with downregulated expression using RNA interference technology
10. PIF4 gene with downregulated expression using RNA interference technology
11. RbcS terminator from Arabidopsis thaliana
12. VTE4 gene from Solanum lycopersicum
13. Chimeric enhancer of the CaMV 35S and HSP18.2 promoters from Arabidopsis thaliana
14. PAP1 gene with upregulated expression using CRISPR/Cas9 gene editing technology
15. Synthetic enhancer Zm13 from Zea mays
16. Spinacia oleracea L-galLDH gene
17. GLK1 gene with downregulated expression using RNAi technology
18. Pea plastocyanin 2A terminator
19. Selectable marker - nptII gene encoding for kanamycin resistance
20. Binary vector for stable genetic transformation
21. ‘Roma’ tomato plant species
This cassette contains the desired genes for increasing the production of lutein, IAA, vitamin E, and vitamin C in tomato leaves while downregulating genes responsible for chlorophyll production and modifying leaf morphology. It also includes regulatory elements like promoters, enhancers, and terminators to ensure high-level gene expression, efficient transcription termination, and prevention of genetic interference. The chosen Agrobacterium-mediated transformation method and binary vector system with a selectable marker will enable stable genetic transformation and selection of transformed tomato plants. Finally, the use of the ‘Roma’ tomato plant species will provide an ideal growth environment for producing edible leaves with improved nutritional content and taste.
**Paper Abstract:** The main objective of this project is to genetically engineer tomato plants to produce edible leaves with higher nutrient content, improved taste, and texture for human consumption. To achieve this goal, we will introduce and/or modify genes involved in the biosynthesis of nutrients such as lutein, vitamin E, vitamin C, and anthocyanins, as well as genes regulating growth and development of leaves. Regulatory elements, including promoters, enhancers, and terminators, will ensure proper expression and termination of the added genes. Agrobacterium-mediated transformation, using a binary vector with selectable markers, will deliver the genetic modifications to the genome of ‘Roma’ tomato plants, an ideal species for mass production of edible leaves. The inclusion of a kanamycin resistance marker will facilitate the identification of transformed plants. The resulting tomato plants will have increased levels of nutrients, improved taste, and texture, making them more appealing and nutritious for human consumption.
**Growth, Selection & Stabilization:** Growth Protocol:
1. Germination: Tomato seeds would be sterilized and planted on sterile soil in a growth chamber at a temperature between 22°C-25°C with a 16:8 h light:dark cycle. The seeds will germinate in about 7-14 days.
2. Plant growth and acclimation: Once the seedlings reach a height of ~5cm, they will be transferred to individual pots containing sterile soil and nutrient-rich media. The tomato plants will be grown under controlled greenhouse conditions, maintaining a temperature of 22°C-25°C with an 18:6 h light: dark cycle. Before being used for experiments, the plants will be acclimated to the greenhouse conditions for at least three weeks to ensure robust growth.
3. Transformation: The binary vector containing the desired genes, regulatory elements, and terminator sequences will be introduced into Agrobacterium tumefaciens strain and cultivated for further use. Tomato plants will be transformed using Agrobacterium tumefaciens.
4. Selection: After Agrobacterium transfer, transformed tomato plants will be selected based on their ability to grow on media containing kanamycin. PCR and gDNA extraction will be performed on selected positive plants to confirm the incorporation of the desired genes and the absence of bacteria or undesired genetic modifications.
5. Growth optimization: The transformed tomato plants will be grown in sterile soil with nutrient-rich media under controlled greenhouse conditions to optimize growth and productivity.
Stabilization Protocol:
1. Genetic stability screening: Plants that pass the initial PCR and gDNA extraction will be bred for several generations (F2 and F3), and the phenotypic stability of the targeted gene expression in the leaves will be evaluated over each generation.
2. Maintenance of environmental conditions: Maintaining consistent greenhouse conditions, temperature, humidity, and light/dark cycles will reduce environmental variables that could affect the phenotype of the tomato plants.
3. Preventing cross-contamination: Preventing cross-contamination with non-transformed tomato plants by isolating the transformed plants physically or using other forms of plant protection.
4. Cryopreservation: Cryopreservation of the transformed tomato plant seeds in long-term storage at very low temperatures or cryobanks would ensure the genetic stability and sustainability of the modified tomato plant lines.
5. Monitoring: Regular monitoring of the transformed tomato plants for any undesirable genetic modifications, and proper testing to ensure the safety and quality of the modified plants for mass consumption.
**Proliferation Method:** After successfully stabilizing the final transformants in tomato plants, the next step would be to proliferate them for mass production of the desired edible leaves. One efficient method for proliferation would be the use of tissue culture techniques.
First, leaf explants would be taken from the transformed tomato plants and sterilized to prevent microbial contamination. These explants would then be cultured on a nutrient-rich medium with proper growth regulators such as cytokinins and auxins to induce cell division and shoot formation.
Once the shoots have grown sufficiently, they can be transferred to a rooting medium to induce root formation. The resulting plantlets can then be transferred to soil and grown in a greenhouse under controlled conditions.
To ensure the maintenance of the desired genetic modifications in the resulting population, a series of quality control measures such as PCR analysis and Southern blotting can be performed to confirm the presence and expression of the added genes. Additionally, the plants can be regularly monitored for any genetic instability or undesirable phenotypic changes.
In conclusion, tissue culture techniques can be used to efficiently proliferate the final transformants in tomato plants and maintain the desired genetic modifications for mass production of edible leaves. Regular quality control measures can ensure genetic stability and the absence of any unwanted phenotypic changes.
**Conclusion:** In conclusion, this project aims to produce tomato plants with higher nutrient content and improved taste and texture by genetically engineering the plants to produce edible leaves with targeted genetic modifications. The transformation protocol described in this project provides a step-by-step guide to achieve successful transformation of the tomato plants using Agrobacterium-mediated transformation with a selectable marker. Further protocols for growth, selection, stabilization, and proliferation of the transformed tomato plants are also described. The gene construct used in this project includes regulatory elements and desired genes for nutrient biosynthesis and improved leaf morphology. The successful production of these genetically modified tomato plants has implications for the agriculture industry in providing more nutrient-rich and desirable produce for human consumption. Future directions for research and development could focus on optimizing growth conditions for the transformed plants, exploring the marketability of these novel plants, and performing further safety and quality testing. Overall, this project serves as a valuable resource for the genetic engineering of tomato plants for improved nutritional value and taste.
As for a pathway from a different species to import, one potential option could be the edible leaf pathways found in some Asian vegetable varieties, such as bok choy or napa cabbage. These species have naturally evolved to produce edible leaves that are not bitter, and have a more desirable texture and flavor for humans. By importing these pathways into tomato plants, it may be possible to stimulate the production of similarly edible and palatable leaves. For example, specific genes involved in the biosynthesis of key compounds responsible for the mild flavor and texture of Asian vegetable leaves could be identified and introduced into the tomato genome.
Overall, the main modifications to investigate in order to achieve the goal of edible tomato leaves for humans would involve targeted gene editing to modify chlorophyll production, as well as the importation of specific genes or pathways responsible for the desirable flavor and texture found in Asian vegetable leaves. It is important to note, however, that ethical considerations must always be taken into account when modifying the genetic makeup of living organisms, and careful testing and regulation should be employed to ensure the safety and environmental impact of any such modifications.
**Genes:** Genes to add:
1. Arabidopsis thaliana LSL1: This gene is involved in the biosynthesis of lutein, a carotenoid pigment that is found in high levels in edible leaves such as spinach and kale. Introducing this gene to tomato plants could increase lutein production in the leaves, which has been shown to have potential health benefits such as reducing the risk of cataracts and age-related macular degeneration.
2. NAPA1: This gene is involved in the biosynthesis of indole-3-acetic acid (IAA), which is a key plant hormone that regulates growth and development. By introducing this gene, it may be possible to stimulate leaf growth and development in tomato plants, leading to the production of larger and more substantial leaves that are easier to harvest and consume.
3. Arabidopsis thaliana DOF1.1: This gene is involved in regulating carbon and nitrogen metabolism in plants, and has been shown to be important for leaf development and morphology. By adding this gene to tomato plants, it may be possible to stimulate the growth of more succulent and tender leaves that are more palatable for human consumption.
4. Solanum lycopersicum VTE4: This gene is involved in the biosynthesis of vitamin E, which is an important nutrient with antioxidant properties. By introducing this gene to tomato plants, it may be possible to increase the amount of vitamin E in the leaves, providing potential health benefits for humans who consume them.
5. Spinacia oleracea L-galLDH: This gene is involved in the biosynthesis of ascorbic acid (vitamin C), which is an important nutrient with antioxidant properties. By introducing this gene to tomato plants, it may be possible to increase the amount of vitamin C in the leaves, providing potential health benefits for humans who consume them.
Genes to tweak:
1. CHLH: This gene is involved in the biosynthesis of chlorophyll, which is responsible for the green color and bitter taste of plant leaves. By using CRISPR/Cas9 gene editing technology to downregulate the expression of this gene in tomato plants, it may be possible to reduce the amount of chlorophyll in the leaves and make them more palatable for human consumption.
2. NOR: This gene is involved in regulating gene expression in response to light. By using RNA interference (RNAi) technology to downregulate the expression of this gene in tomato plants, it may be possible to stimulate leaf growth and development, leading to the production of more substantial leaves that are easier to harvest and consume.
3. PIF4: This gene is involved in regulating plant growth and development in response to light. By using RNAi technology to downregulate the expression of this gene in tomato plants, it may be possible to stimulate leaf growth and development, leading to the production of more substantial leaves that are easier to harvest and consume.
4. PAP1: This gene is involved in regulating the biosynthesis of anthocyanin pigments that give plants their purple or red coloration. By using CRISPR/Cas9 gene editing technology to upregulate the expression of this gene in tomato plants, it may be possible to increase the production of anthocyanins in the leaves, potentially providing health benefits and making the leaves more attractive for human consumption.
5. GLK1: This gene is involved in regulating the expression of genes involved in photosynthesis, and has been shown to play a role in leaf morphology and development. By using RNAi technology to downregulate the expression of this gene in tomato plants, it
**Regulatory Elements:** may be possible to alter leaf morphology and make them more tender and palatable for human consumption. Additionally, downregulation of this gene may also result in the reduction of chlorophyll in the leaves, further improving their taste and texture.
Promoters:
1. The ubiquitin promoter from Solanum lycopersicum: This promoter has been shown to be highly active in many parts of tomato plants, including leaves. Using this promoter to drive the expression of the added genes, such as LSL1 and NAPA1, may ensure high levels of gene expression in the leaves, leading to increased lutein and IAA production and possibly larger and more succulent leaves.
2. The 35S promoter from Cauliflower mosaic virus: This promoter is commonly used in genetic engineering projects and has been shown to be highly active in many different plant species. Using this promoter to drive the expression of the added genes, such as DOF1.1 and VTE4, may ensure high levels of gene expression in the leaves, leading to increased vitamin E production and possibly more palatable and healthy leaves.
3. The CaMV 35S promoter variant from Solanum chacoense: This promoter variant has been shown to be highly active in tomato plants, including leaves. Using this promoter to drive the expression of the added genes, such as Spinacia oleracea L-galLDH and PAP1, may ensure high levels of gene expression in the leaves, leading to increased vitamin C and anthocyanin production and potentially more appealing and nutritious leaves.
Enhancers:
1. The synthetic enhancer Zm13 from Zea mays: This enhancer has been shown to enhance gene expression in various plant species, including tomato. Using this enhancer in combination with the chosen promoters may further boost gene expression levels in the tomato leaves, leading to desired outcomes such as increased lutein, vitamin E, and vitamin C production.
2. The chimeric enhancer of the CaMV 35S and HSP18.2 promoters from Arabidopsis thaliana: This enhancer has been shown to be active in various plant tissues and can enhance gene expression levels when used in combination with some promoters. Using this enhancer in combination with the chosen promoters may stimulate leaf growth and development, leading to the production of more substantial and palatable leaves.
3. The chloroplast transit peptide enhancer from Solanum lycopersicum: This enhancer has been shown to enhance the expression of genes targeted to the chloroplast, the site of photosynthesis in plant cells. Using this enhancer in conjunction with the chosen promoters may increase the efficiency of the added genes in chloroplasts and result in increased production of nutrients such as lutein and vitamin E in the tomato leaves.
Terminators:
1. The nos terminator from Agrobacterium tumefaciens: This terminator sequence is commonly used in genetic engineering applications and has been shown to be effective in many plant species. Using this terminator to finish the transcription of the added genes in the tomato leaves may ensure that the genes are expressed at the intended levels and do not interfere with other plant processes.
2. The rbcS terminator from Arabidopsis thaliana: This terminator sequence is derived from the gene encoding ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), a key enzyme in photosynthesis. Using this terminator in combination with the chosen promoters may result in more efficient expression of the added genes in the tomato leaves, as it is derived from a gene commonly expressed in leaves.
3. The pea plastocyan
**Vector & Delivery:** in 2A terminator: This terminator sequence is derived from the gene encoding plastocyanin, a key protein involved in electron transfer during photosynthesis. Using this terminator in conjunction with the chosen promoters may enhance the expression of the added genes in chloroplasts and result in increased production of nutrients such as lutein and vitamin E in the tomato leaves. Additionally, this terminator may ensure proper termination of transcription and prevent interference with other plant processes.
Vector:
I would choose the Agrobacterium-mediated transformation method to introduce the desired genetic modifications in tomato plants. Agrobacterium tumefaciens is a soil bacterium that has been shown to transfer DNA to plant cells via a natural process, making it a useful tool in genetic engineering. The advantage of this method is that Agrobacterium can deliver large pieces of DNA to the plant genome, allowing for the efficient transfer of multiple genes at once. The use of Agrobacterium as a vector system also eliminates the need for particle bombardment or electroporation, which can damage plant cells and reduce transformation efficiency.
For this particular project, I would use a binary vector containing the desired genes, regulatory elements, and terminator sequences, along with selectable markers for identifying and selecting transformed plants. The binary vector would also contain the T-DNA borders required for integration into the plant genome via Agrobacterium transfer. The use of a binary vector enhances the stability and versatility of the system, enabling the introduction of multiple genes and the ability to screen for transformants using selectable markers.
Species:
The tomato plant variety ‘Roma’ would be an ideal choice for this project as it is a widely cultivated variety with a compact growth habit and a relatively short lifespan, making it easier to manipulate for transformation and experimentation. ‘Roma’ also has a high yield potential, making it suitable for mass production of edible leaves. Finally, ‘Roma’ is a diploid species, meaning it has two sets of chromosomes, making it easier to manipulate genetically compared to polyploid species.
In conclusion, the use of Agrobacterium-mediated transformation with a binary vector containing the desired genes, regulatory elements, and terminator sequences would optimize the genetic modifications desired for creating edible tomato leaves. The choice of ‘Roma’ tomato plant as the species would provide an ideal growth environment, making it easier to manipulate and mass-produce edible leaves containing lutein, vitamin E, vitamin C, and anthocyanin.
**Selection Marker:** If a selection marker is deemed needed for this project we will use the nptII gene encoding for kanamycin resistance. This marker is commonly used in Agrobacterium-mediated genetic transformation, and it allows for the selection of transformed plants through the use of kanamycin-containing media. The successful transformation and integration of the T-DNA containing the desired genetic modifications into the tomato genome will confer kanamycin resistance, allowing for the selection of transformed plants while avoiding the use of antibiotic resistance markers. The inclusion of a selection marker will facilitate the identification of successfully modified tomato plants, increasing the efficiency of the transformation process and ensuring the desired phenotypic changes are present in the resulting tomato plants.
**Transformation Protocol:** Protocol for transforming tomato plants with desired genetic modifications:
Materials:
- Tomato ‘Roma’ plantlets (preferably in vitro propagated)
- Agrobacterium tumefaciens strain suitable for the vectors being used
- Binary vector containing desired genes, regulatory elements, terminator sequences, and selectable markers
- LB agar plates (with appropriate antibiotics)
- Sterile NaCl solution (0.9%)
- Sterile water
- Murashige and Skoog (MS) medium (with appropriate antibiotics)
- Kanamycin (if using selection marker)
- Sterile filter-tips and pipettes
- Sterilized forceps and scalpel
- Growth chamber/ greenhouse
Procedure:
1. Culture the Agrobacterium strain with the binary vector on an LB agar plate with appropriate antibiotics and incubate overnight at 28°C.
2. Inoculate a single colony from the overnight culture of Agrobacterium in 5 ml of LB medium with appropriate antibiotics and incubate for 16-24 hours at 28°C with constant shaking.
3. Wash the Agrobacterium cells twice in a sterile NaCl solution (0.9%) by centrifuging at 3000 rpm for 10 minutes and resuspending in the same solution.
4. In a new culture tube, mix 200 µl of the Agrobacterium suspension with 2 ml of MS medium containing appropriate antibiotics and 150 µM acetosyringone. Shake gently and let it incubate at room temperature for at least 3 hours.
5. Sterilize the tomato plantlets by washing them in sterile water for 10 minutes, followed by a 15-minute treatment with 70% ethanol and rinsing them 3 times in sterile water.
6. Collect the sterilized tomato plantlets with sterilized forceps and scalpel and cut off the cotyledons and hypocotyls.
7. Transfer the plantlets onto a new MS medium plate containing appropriate antibiotics and wait for 2-3 days for the plantlets to recover.
8. Take 3-4 fully expanded leaves from the plantlets for transformation (avoid using the first two leaves) and throw away the rest of the plantlet.
9. Using a wide-bore pipette tip, puncture the abaxial surface of the tomato leaves to create small wounds without damaging the veins.
10. Dip the wounded tomato leaves into the Agrobacterium suspension, taking care to ensure that the suspension completely covers the wounded area of the tomato leaves.
11. Co-cultivate the tomato leaves with Agrobacterium in the dark at 25°C for 2-3 days.
12. After co-cultivation, place the tomato leaves onto a new MS medium plate containing appropriate antibiotics and incubate for 2-3 weeks in the growth chamber/greenhouse.
13. Transfer the successfully transformed tomato leaves to a new MS medium plate containing kanamycin (if using selection marker) and wait for 2-3 weeks for the formation of new shoots.
14. When new shoots are 1-2cm long, transfer them to fresh MS medium plates containing appropriate antibiotics and wait for the plantlets to grow.
15. Once the plantlets have reached a suitable size, they can be transferred to soil for further growth and experimentation.
By following this protocol, tomato plants can be successfully transformed with the desired genetic modifications using Agrobacterium-mediated transformation. The inclusion of a selection marker (if needed) further facilitates the identification of successfully modified tomato plants, increasing the efficiency of the transformation process and ensuring the desired phenotypic changes are present in
**Gene cassette**: The 5' to 3' list of elements in the multi-purpose cassette are as follows:
1. Ubiquitin promoter from Solanum lycopersicum
2. LSL1 gene from Arabidopsis thaliana
3. NAPA1 gene
4. 35S promoter from Cauliflower mosaic virus
5. DOF1.1 gene from Arabidopsis thaliana
6. Nos terminator from Agrobacterium tumefaciens
7. CHLH gene with downregulated expression using CRISPR/Cas9 gene editing technology
8. Zm13 enhancer from Zea mays
9. NOR gene with downregulated expression using RNA interference technology
10. PIF4 gene with downregulated expression using RNA interference technology
11. RbcS terminator from Arabidopsis thaliana
12. VTE4 gene from Solanum lycopersicum
13. Chimeric enhancer of the CaMV 35S and HSP18.2 promoters from Arabidopsis thaliana
14. PAP1 gene with upregulated expression using CRISPR/Cas9 gene editing technology
15. Synthetic enhancer Zm13 from Zea mays
16. Spinacia oleracea L-galLDH gene
17. GLK1 gene with downregulated expression using RNAi technology
18. Pea plastocyanin 2A terminator
19. Selectable marker - nptII gene encoding for kanamycin resistance
20. Binary vector for stable genetic transformation
21. ‘Roma’ tomato plant species
This cassette contains the desired genes for increasing the production of lutein, IAA, vitamin E, and vitamin C in tomato leaves while downregulating genes responsible for chlorophyll production and modifying leaf morphology. It also includes regulatory elements like promoters, enhancers, and terminators to ensure high-level gene expression, efficient transcription termination, and prevention of genetic interference. The chosen Agrobacterium-mediated transformation method and binary vector system with a selectable marker will enable stable genetic transformation and selection of transformed tomato plants. Finally, the use of the ‘Roma’ tomato plant species will provide an ideal growth environment for producing edible leaves with improved nutritional content and taste.
**Paper Abstract:** The main objective of this project is to genetically engineer tomato plants to produce edible leaves with higher nutrient content, improved taste, and texture for human consumption. To achieve this goal, we will introduce and/or modify genes involved in the biosynthesis of nutrients such as lutein, vitamin E, vitamin C, and anthocyanins, as well as genes regulating growth and development of leaves. Regulatory elements, including promoters, enhancers, and terminators, will ensure proper expression and termination of the added genes. Agrobacterium-mediated transformation, using a binary vector with selectable markers, will deliver the genetic modifications to the genome of ‘Roma’ tomato plants, an ideal species for mass production of edible leaves. The inclusion of a kanamycin resistance marker will facilitate the identification of transformed plants. The resulting tomato plants will have increased levels of nutrients, improved taste, and texture, making them more appealing and nutritious for human consumption.
**Growth, Selection & Stabilization:** Growth Protocol:
1. Germination: Tomato seeds would be sterilized and planted on sterile soil in a growth chamber at a temperature between 22°C-25°C with a 16:8 h light:dark cycle. The seeds will germinate in about 7-14 days.
2. Plant growth and acclimation: Once the seedlings reach a height of ~5cm, they will be transferred to individual pots containing sterile soil and nutrient-rich media. The tomato plants will be grown under controlled greenhouse conditions, maintaining a temperature of 22°C-25°C with an 18:6 h light: dark cycle. Before being used for experiments, the plants will be acclimated to the greenhouse conditions for at least three weeks to ensure robust growth.
3. Transformation: The binary vector containing the desired genes, regulatory elements, and terminator sequences will be introduced into Agrobacterium tumefaciens strain and cultivated for further use. Tomato plants will be transformed using Agrobacterium tumefaciens.
4. Selection: After Agrobacterium transfer, transformed tomato plants will be selected based on their ability to grow on media containing kanamycin. PCR and gDNA extraction will be performed on selected positive plants to confirm the incorporation of the desired genes and the absence of bacteria or undesired genetic modifications.
5. Growth optimization: The transformed tomato plants will be grown in sterile soil with nutrient-rich media under controlled greenhouse conditions to optimize growth and productivity.
Stabilization Protocol:
1. Genetic stability screening: Plants that pass the initial PCR and gDNA extraction will be bred for several generations (F2 and F3), and the phenotypic stability of the targeted gene expression in the leaves will be evaluated over each generation.
2. Maintenance of environmental conditions: Maintaining consistent greenhouse conditions, temperature, humidity, and light/dark cycles will reduce environmental variables that could affect the phenotype of the tomato plants.
3. Preventing cross-contamination: Preventing cross-contamination with non-transformed tomato plants by isolating the transformed plants physically or using other forms of plant protection.
4. Cryopreservation: Cryopreservation of the transformed tomato plant seeds in long-term storage at very low temperatures or cryobanks would ensure the genetic stability and sustainability of the modified tomato plant lines.
5. Monitoring: Regular monitoring of the transformed tomato plants for any undesirable genetic modifications, and proper testing to ensure the safety and quality of the modified plants for mass consumption.
**Proliferation Method:** After successfully stabilizing the final transformants in tomato plants, the next step would be to proliferate them for mass production of the desired edible leaves. One efficient method for proliferation would be the use of tissue culture techniques.
First, leaf explants would be taken from the transformed tomato plants and sterilized to prevent microbial contamination. These explants would then be cultured on a nutrient-rich medium with proper growth regulators such as cytokinins and auxins to induce cell division and shoot formation.
Once the shoots have grown sufficiently, they can be transferred to a rooting medium to induce root formation. The resulting plantlets can then be transferred to soil and grown in a greenhouse under controlled conditions.
To ensure the maintenance of the desired genetic modifications in the resulting population, a series of quality control measures such as PCR analysis and Southern blotting can be performed to confirm the presence and expression of the added genes. Additionally, the plants can be regularly monitored for any genetic instability or undesirable phenotypic changes.
In conclusion, tissue culture techniques can be used to efficiently proliferate the final transformants in tomato plants and maintain the desired genetic modifications for mass production of edible leaves. Regular quality control measures can ensure genetic stability and the absence of any unwanted phenotypic changes.
**Conclusion:** In conclusion, this project aims to produce tomato plants with higher nutrient content and improved taste and texture by genetically engineering the plants to produce edible leaves with targeted genetic modifications. The transformation protocol described in this project provides a step-by-step guide to achieve successful transformation of the tomato plants using Agrobacterium-mediated transformation with a selectable marker. Further protocols for growth, selection, stabilization, and proliferation of the transformed tomato plants are also described. The gene construct used in this project includes regulatory elements and desired genes for nutrient biosynthesis and improved leaf morphology. The successful production of these genetically modified tomato plants has implications for the agriculture industry in providing more nutrient-rich and desirable produce for human consumption. Future directions for research and development could focus on optimizing growth conditions for the transformed plants, exploring the marketability of these novel plants, and performing further safety and quality testing. Overall, this project serves as a valuable resource for the genetic engineering of tomato plants for improved nutritional value and taste.