Create garlic that could be dried and popped like popcorn - Your Published Bio Team Output
**Pre-Project**To achieve the objective of creating garlic that could be dried and popped like popcorn, we would need to modify the endogenous sulfur metabolism pathway that is responsible for the pungent odor and taste of garlic. The modification should be such that the production of volatile sulfur compounds is reduced as it is these compounds that release the pungent smell and taste of garlic.
As for a pathway to import, we can consider the pathway for the production of an antioxidant called anthocyanin from the blue corn maize plant. This pathway is known to produce stable pigments, which makes it an ideal pathway to import into garlic. By importing this pathway into garlic, we can enhance its shelf life and make it more resistant to damage during storage.
To achieve these modifications, we would need to invest in gene editing technology and genetic engineering. By using CRISPR Cas-9 gene editing technology, we can selectively edit the genes involved in the sulfur metabolism pathway and reduce the production of volatile sulfur compounds. We can also use transformation techniques to import the anthocyanin pathway from blue corn maize plant into garlic.
Overall, this modification would support the food industry by increasing the shelf life of garlic and allowing for greater culinary use of the garlic as a seasoning pop like popcorn. Additionally, it could have health benefits as garlic is packed with antioxidants, vitamins and minerals, and the production of volatile sulfur compounds can prevent people from eating enough of this healthy food.
**Genes:** 1. Alliinase gene: Alliinase is an enzyme that plays a key role in garlic's sulfur metabolism pathway, converting the sulfur-containing compound alliin to allicin, which is the main source of garlic's pungent odor and taste. By using CRISPR Cas-9 gene editing technology to knock out or decrease the expression of the alliinase gene, we can reduce the amount of allicin produced in garlic and thus decrease its pungent taste and odor.
2. Beta-carotene hydroxylase gene: Beta-carotene hydroxylase is an enzyme that is involved in the production of vitamin A, an essential nutrient that is lacking in many people's diets. By importing the beta-carotene hydroxylase gene from other plants into garlic, we can enhance the production of vitamin A in garlic and provide a new source of this important nutrient.
3. Anthocyanin pathway genes: As mentioned in the pre-genes, importing the anthocyanin pathway genes from blue corn maize into garlic can enhance its shelf-life and make it more resistant to damage during storage. This pathway includes several genes, including dihydroflavonol reductase, flavonoid 3',5'-hydroxylase, and anthocyanidin synthase.
4. Superoxide dismutase gene: Superoxide dismutase is an enzyme that plays a crucial role in scavenging harmful reactive oxygen species (ROS) in cells. By overexpressing the superoxide dismutase gene in garlic, we can increase the level of ROS scavenging activity and thus enhance the antioxidant capacity of garlic.
5. Invertase gene: Invertase is an enzyme that plays a key role in sucrose metabolism. By increasing the expression level of the invertase gene in garlic, we can enhance its sugar metabolism pathway and potentially increase the sugar content of garlic, which could make it more suitable for popping like popcorn.
**Regulatory Elements:** Ideal Promoter Sequences:
1. CaMV 35S Promoter - This promoter sequence is commonly used in plant genetic engineering and is known to be highly active in various plant tissues. This promoter can be used to drive the expression of the beta-carotene hydroxylase gene in garlic, which could enhance its vitamin A production.
2. OinC Promoter - This promoter sequence is known to be active in Allium species such as onions and garlic. It can be used to drive the expression of the alliinase gene in garlic, which can reduce the pungent taste and odor of garlic.
3. Zein Promoter - This promoter sequence is known to be active in maize and can be used to drive the expression of the anthocyanin pathway genes in garlic, which could enhance its shelf-life and make it more resistant to damage during storage.
Ideal Enhancer Sequences:
1. CaMV 35S Enhancer - This enhancer sequence can enhance the activity of the CaMV 35S promoter used to drive the beta-carotene hydroxylase gene in garlic, resulting in higher vitamin A production.
2. 5' upstream sequence of the Alliinase gene - This enhancer sequence can enhance the expression of the alliinase gene in garlic, which can reduce its pungent taste and odor.
3. PAL Enhancer - Phenylalanine ammonia-lyase (PAL) is an enzyme involved in the anthocyanin pathway. The PAL enhancer sequence can enhance the expression of PAL and subsequently enhance the expression of the anthocyanin pathway genes in garlic, which could improve its shelf-life.
Ideal Terminator Sequences:
1. Nos Terminator - This terminator sequence is commonly used in plant genetic engineering and is known to function efficiently in various plant tissues.
2. T35S Terminator - This terminator sequence is derived from the CaMV 35S promoter and can be used to terminate the expression of the beta-carotene hydroxylase gene in garlic.
3. NOS Polyadenylation Signal - This sequence is derived from the nopaline synthase (NOS) gene and is commonly used as a terminator sequence in plant genetic engineering. It can be used to terminate the expression of the alliinase, anthocyanin pathway, superoxide dismutase, and invertase genes in garlic.
**Vector & Delivery:** Vector: The Agrobacterium tumefaciens binary vector pCAMBIA 1300 will be an appropriate vector for delivering the desired genetic modifications into garlic plants. This vector has a broad host range, including Allium species, and allows the introduction of multiple genes at the same time. The pCAMBIA 1300 vector also carries a plant selectable marker (selection gene), which confers resistance to kanamycin or hygromycin. Selection gene is used to identify transformed cells and plants, which will facilitate the identification of garlic plants that have undergone the desired genetic modifications.
Delivery and Modification Method: Agrobacterium-mediated transformation will be the method of choice for delivering the desired genes into garlic plants. This method involves using a naturally occurring soil bacterium, Agrobacterium tumefaciens, as a delivery vehicle for transferring genes into plant cells. The bacterium is engineered to carry a modified plasmid that contains the desired genes under the control of the regulatory elements described above. The garlic plants are then infected with the engineered Agrobacterium, resulting in the transfer of the desired genes into the plant cells.
The infected cells are grown in selective media containing kanamycin or hygromycin, which kills cells that did not incorporate the selection gene. Transformed cells will grow and form calli, which can then be screened for the desired genetic modifications using PCR and other molecular biology methods. Transformed calli are then induced to regenerate into whole plants, which can be transferred to soil for further growth and evaluation.
Agrobacterium-mediated transformation is a widely used and effective method for delivering genes into plants, including garlic. Compared to other methods such as biolistics, it has a higher transformation efficiency and a lower risk of inducing random mutations or damage to the plant genome. Therefore, it is an appropriate method for achieving the desired genetic modifications in garlic plants efficiently and with high precision.
**Selection Marker:** If a selection marker is deemed needed for this project, we will use a kanamycin or hygromycin selectable marker carried on the pCAMBIA 1300 vector. This will facilitate the identification of transformed garlic plants that have incorporated the desired genetic modifications. The selectable marker will confer resistance to either kanamycin or hygromycin, allowing for the growth and selection of transformed cells on culture media containing the corresponding antibiotic. Functionally, this will make it easy to differentiate transformed cells from untransformed cells in culture, making it easier to screen and select transformed cells for further growth and analysis. Furthermore, the use of a selectable marker that is different from garlic's native antibiotic resistance will reduce the risk of horizontal gene transfer to opportunistic pathogenic bacteria, thus improving biosafety.
**Transformation Protocol:** Protocol for transforming garlic with selected genes:
Materials:
- Garlic bulbs for transformation
- Agrobacterium tumefaciens binary vector pCAMBIA 1300
- Antibiotics for selection (kanamycin or hygromycin)
- Murashige and Skoog (MS) medium
- Plant growth hormones (auxins and cytokinins)
- Sterilized instruments (scissors, forceps, scalpels)
- Sterile Petri dishes
- Culture flasks
- PCR primers for gene detection
- PCR machine
- DNA extraction kit
- Gel electrophoresis equipment
- Ethidium bromide stain
Steps:
1. Preparation of Agrobacterium tumefaciens with the desired plasmid:
- Grow Agrobacterium tumefaciens on a selective medium containing kanamycin or hygromycin overnight.
- Harvest the bacterial culture by centrifugation at 4000 rpm for 15 minutes.
- Resuspend the bacterial pellet in sterilized water or buffer.
2. Garlic bulbs surface sterilization and explant preparation:
- Clean the surface of the garlic bulb with 70% ethanol.
- Peel off the outermost layer of the garlic bulb to expose the cloves.
- Cut the cloves into small explants, approximately 5 mm in size.
- Place the explants in a sterile Petri dish.
3. Co-cultivation of garlic explants with Agrobacterium:
- Mix the Agrobacterium suspension with the garlic explants in a sterile environment, incubate at 25°C for 2-3 days, and enable co-cultivation.
- Carefully remove the explants from the suspension and place them onto a sterile MS medium with antibiotics (kanamycin or hygromycin).
4. Selection and regeneration of transformed cells:
- Culture the explants on a selection medium containing kanamycin or hygromycin, making sure to screen out non-transformed cells.
- Incubate the Petri dishes under controlled temperature and light conditions (25°C, 16h light/8h dark cycle).
- After 2-3 weeks, monitor the explants for the presence of calli, growing masses of undifferentiated cells.
- Identify calli which display the desired genetic modifications using PCR and electrophoresis of PCR products.
- Induce the regeneration of transformed cells by transferring them to a MS medium containing appropriate concentrations of auxins and cytokinins to encourage plant development.
5. Transplanting and acclimatization of transformed plants:
- After one month, transfer the plantlets to soil or a suitable substrate and grow them under controlled conditions (temperature, humidity, light).
- After acclimatization, transfer the plants to the field for further growth and evaluation of phenotypic traits.
6. Characterization of the transformed plants:
- Verify the genetic modifications in the regenerated plants using PCR and electrophoresis of DNA isolated from the leaves.
- Quantify the expression levels of the target genes through RT-PCR.
- Evaluate the phenotype of transformed garlic plants, examining traits such as allicin content, vitamin A content and antioxidant capacity.
- Compare phenotypes and genetic modifications between transformed and non-transformed groups to observe the impact of the desired genetic modifications on garlic's traits.
Note: Biosafety protocols must be followed to ensure that the transformed garlic plants do not present any environmental or health risks.
**Gene cassette**: The 5' to 3' list of elements in the multi-purpose cassette are as follows:
1. CaMV 35S Promoter - to drive the expression of the Beta-carotene hydroxylase gene for enhanced vitamin A production.
2. Beta-carotene hydroxylase gene - imported from other plants into garlic for enhanced production of vitamin A.
3. CaMV 35S Enhancer - to enhance the activity of the CaMV 35S promoter used to drive the beta-carotene hydroxylase gene.
4. T35S Terminator - derived from the CaMV 35S promoter and can be used to terminate the expression of the beta-carotene hydroxylase gene in garlic.
5. OinC Promoter - to drive the expression of the Alliinase gene for reducing garlic's pungent taste and odor.
6. 5' upstream sequence of the Alliinase gene - to enhance the expression of the Alliinase gene in garlic.
7. NOS Polyadenylation Signal - to terminate the expression of the Alliinase, Anthocyanin pathway genes, Superoxide dismutase, and Invertase genes in garlic.
8. Anthocyanin pathway genes - Dihydroflavonol reductase, Flavonoid 3',5'-hydroxylase, and Anthocyanidin synthase for enhanced garlic shelf-life and resistance to damage during storage.
9. PAL Enhancer - to enhance the expression of Phenylalanine ammonia-lyase and, subsequently, enhance the expression of the anthocyanin pathway genes in garlic.
10. Superoxide dismutase gene - to increase the level of ROS scavenging activity and enhance the antioxidant capacity of garlic.
11. Invertase gene - to enhance the sugar metabolism pathway and potentially increase the sugar content of garlic.
12. Nos Terminator - commonly used in plant genetic engineering and is known to function efficiently in various plant tissues to terminate the expression of the desired genes.
13. pCAMBIA 1300 Vector - a broad host range vector that allows the introduction of multiple genes at the same time and carries a plant selectable marker for identification of transformed garlic plants.
14. Agrobacterium-mediated transformation - an effective method for delivering genes into garlic plants with a higher transformation efficiency and a lower risk of inducing random mutations or damage to the plant genome.
15. Kanamycin or Hygromycin Selectable Marker - used to differentiate transformed cells from untransformed cells in culture and reduce the risk of horizontal gene transfer to opportunistic pathogenic bacteria, improving biosafety.
**Paper Abstract:** The main objective of this study is to use CRISPR Cas-9 gene editing technology and gene insertion techniques to modify garlic by introducing or knocking out genes associated with its nutritional and sensory properties. Specifically, we aim to reduce the pungency of garlic by knocking out or reducing the expression of the Alliinase gene, enhance its vitamin A production by inserting the Beta-carotene hydroxylase gene, improve its shelf-life by introducing anthocyanin pathway genes from blue corn maize, increase its antioxidant capacity by overexpressing the Superoxide dismutase gene, and enhance its sugar metabolism pathway by increasing the expression level of the Invertase gene. To achieve these objectives, we will use regulatory elements such as ideal promoter, enhancer, and terminator sequences that are active in garlic and other compatible plant species. We will deliver the desired genes into garlic plants using Agrobacterium-mediated transformation, a widely used and highly efficient method for delivering genes into plant cells.
The implications of this study are significant as it opens up new possibilities for modifying the nutritional and sensory properties of garlic. Specifically, the development of low-pungency, high vitamin A, and improved shelf-life garlic could have significant health benefits for consumers, especially those with vitamin A deficiency or who cannot consume highly pungent garlic due to health conditions. Furthermore, garlic could become a viable source of vitamin A, which is usually obtained from fruits and vegetables that are not always readily available or affordable in certain regions. The enhanced sugar content in garlic could also have implications for its use as a snack or culinary ingredient. Overall, the findings of this study could pave the way for further research on modifying other plant species to enhance their nutritional and sensory properties.
**Growth, Selection & Stabilization:** Growth and Selection Protocol:
1. Garlic plant growth conditions:
- Garlic plants should be grown in well-draining soil with a pH of 6.0-7.5 and adequate moisture.
- Temperature: 15-25°C
- Light: Garlic plants should be exposed to full sunlight for at least 6 hours per day.
- Nutrients: Garlic plants should be fertilized with a balanced fertilizer containing nitrogen, phosphorus, and potassium. Additional fertilization with micronutrients may be needed depending on soil conditions.
- Growth stage: Garlic plants should be grown until they reach the vegetative stage, approximately 6-8 weeks after planting.
2. Agrobacterium culture conditions:
- Agrobacterium tumefaciens strain carrying the desired plasmid should be grown in LB (Luria-Bertani) media supplemented with appropriate antibiotics (e.g. rifampicin) at 28°C with agitation.
3. Delivery of the desired genes into garlic plants:
- The garlic plants should be injured by cutting their basal plate with a scalpel to facilitate infection by the Agrobacterium.
- The Agrobacterium cells carrying the desired genes should be added to the garlic plant wound induced on the cutting plate.
- Infected garlic plants should be incubated in a humid environment for 48 hours to allow for Agrobacterium attachment and gene transfer.
4. Selection of transformed cells:
- Infected garlic plants should be transferred to a selection medium containing kanamycin (50 mg/L) or hygromycin (20 mg/L).
- Calli generated from the infected cells should be screened for the presence of the desired genetic modifications using PCR and sequencing. It is advisable to obtain a minimum of three regenerated plants for all gene-lines.
- Transformed calli are then induced to regenerate into whole plants, which can then be transferred to soil for further growth and evaluation.
5. Stabilization of the modified organisms:
- Transgenic garlic plants should be crossed with wild-type garlic plants to increase the genetic diversity and minimize the risk of unintended consequences.
- Stable lines of the modified garlic plants should be maintained in isolated fields, in separated greenhouses or by other physical means, preventing cross-pollination with non-GM garlic and minimizing gene flow.
- Regular monitoring for the presence of the introduced genes and their phenotypic consequences should be undertaken through regular use of PCR and phenotypic analysis under controlled conditions.
**Proliferation Method:** To proliferate the final transformants once stabilized, we will use tissue culture techniques to multiply the plants. Specifically, we will use a method known as micropropagation, which involves growing plant tissue on agar media in aseptic conditions. Individual garlic plants can be multiplied into large numbers of identical clones using this method.
To efficiently proliferate the desired genetic modifications in the resulting population, we will ensure that all the transformed plants display stable inheritance of the introduced genetic modifications. We will confirm this stability by conducting PCR analysis and other molecular biology methods at different stages of plant growth and development, including after callus formation, plant regeneration, and during the early stages of growth in soil.
To maintain the desired genetic modifications, we will avoid any cross-pollination or hybridization between the transformed garlic plants and other garlic plants in the vicinity. We will take measures such as growing the plants in a confined environment or using physical barriers to ensure that the transformed garlic plants do not cross with any other garlic plants. We will also isolate the plants that display the desired genetic modifications and grow them separately to avoid any unintended gene flow.
In summary, by using tissue culture techniques such as micropropagation, and taking measures to ensure genetic stability and prevent cross-pollination or hybridization, we can efficiently proliferate the desired genetic modifications in garlic plants and maintain these modifications in the resulting population for further study or commercialization.
**Conclusion:** In conclusion, the project aimed to modify garlic through genetic engineering techniques. The first protocol outlined the steps to introduce desired genes into garlic, using Agrobacterium-mediated transformation. After selection and regeneration of transformed cells, stable lines of the modified garlic plants were to be maintained to evaluate the phenotypic traits. The second protocol aimed to proliferate the final transformants once stabilized and maintain the desired genetic modifications to efficiently proliferate the desired genetic modifications. The third protocol suggested constructing a multi-purpose cassette with a list of elements that could modify garlic by reducing pungency, enhancing vitamin A production, improving shelf-life, increasing antioxidant capacity and sugar content. The significance of this study is significant, as the modifications could have considerable benefits for consumers and the agriculture industry. The study could pave the way for further research on developing other plant species to enhance their nutritional and sensory properties. However, biosafety protocols must be followed to ensure that these modified garlic plants do not pose any health or environmental hazards. Overall, the project has been successful in outlining protocols for garlic transformation, growth and selection, and proliferation, which could open new possibilities for modifying the nutritional and sensory properties of other plant species.
As for a pathway to import, we can consider the pathway for the production of an antioxidant called anthocyanin from the blue corn maize plant. This pathway is known to produce stable pigments, which makes it an ideal pathway to import into garlic. By importing this pathway into garlic, we can enhance its shelf life and make it more resistant to damage during storage.
To achieve these modifications, we would need to invest in gene editing technology and genetic engineering. By using CRISPR Cas-9 gene editing technology, we can selectively edit the genes involved in the sulfur metabolism pathway and reduce the production of volatile sulfur compounds. We can also use transformation techniques to import the anthocyanin pathway from blue corn maize plant into garlic.
Overall, this modification would support the food industry by increasing the shelf life of garlic and allowing for greater culinary use of the garlic as a seasoning pop like popcorn. Additionally, it could have health benefits as garlic is packed with antioxidants, vitamins and minerals, and the production of volatile sulfur compounds can prevent people from eating enough of this healthy food.
**Genes:** 1. Alliinase gene: Alliinase is an enzyme that plays a key role in garlic's sulfur metabolism pathway, converting the sulfur-containing compound alliin to allicin, which is the main source of garlic's pungent odor and taste. By using CRISPR Cas-9 gene editing technology to knock out or decrease the expression of the alliinase gene, we can reduce the amount of allicin produced in garlic and thus decrease its pungent taste and odor.
2. Beta-carotene hydroxylase gene: Beta-carotene hydroxylase is an enzyme that is involved in the production of vitamin A, an essential nutrient that is lacking in many people's diets. By importing the beta-carotene hydroxylase gene from other plants into garlic, we can enhance the production of vitamin A in garlic and provide a new source of this important nutrient.
3. Anthocyanin pathway genes: As mentioned in the pre-genes, importing the anthocyanin pathway genes from blue corn maize into garlic can enhance its shelf-life and make it more resistant to damage during storage. This pathway includes several genes, including dihydroflavonol reductase, flavonoid 3',5'-hydroxylase, and anthocyanidin synthase.
4. Superoxide dismutase gene: Superoxide dismutase is an enzyme that plays a crucial role in scavenging harmful reactive oxygen species (ROS) in cells. By overexpressing the superoxide dismutase gene in garlic, we can increase the level of ROS scavenging activity and thus enhance the antioxidant capacity of garlic.
5. Invertase gene: Invertase is an enzyme that plays a key role in sucrose metabolism. By increasing the expression level of the invertase gene in garlic, we can enhance its sugar metabolism pathway and potentially increase the sugar content of garlic, which could make it more suitable for popping like popcorn.
**Regulatory Elements:** Ideal Promoter Sequences:
1. CaMV 35S Promoter - This promoter sequence is commonly used in plant genetic engineering and is known to be highly active in various plant tissues. This promoter can be used to drive the expression of the beta-carotene hydroxylase gene in garlic, which could enhance its vitamin A production.
2. OinC Promoter - This promoter sequence is known to be active in Allium species such as onions and garlic. It can be used to drive the expression of the alliinase gene in garlic, which can reduce the pungent taste and odor of garlic.
3. Zein Promoter - This promoter sequence is known to be active in maize and can be used to drive the expression of the anthocyanin pathway genes in garlic, which could enhance its shelf-life and make it more resistant to damage during storage.
Ideal Enhancer Sequences:
1. CaMV 35S Enhancer - This enhancer sequence can enhance the activity of the CaMV 35S promoter used to drive the beta-carotene hydroxylase gene in garlic, resulting in higher vitamin A production.
2. 5' upstream sequence of the Alliinase gene - This enhancer sequence can enhance the expression of the alliinase gene in garlic, which can reduce its pungent taste and odor.
3. PAL Enhancer - Phenylalanine ammonia-lyase (PAL) is an enzyme involved in the anthocyanin pathway. The PAL enhancer sequence can enhance the expression of PAL and subsequently enhance the expression of the anthocyanin pathway genes in garlic, which could improve its shelf-life.
Ideal Terminator Sequences:
1. Nos Terminator - This terminator sequence is commonly used in plant genetic engineering and is known to function efficiently in various plant tissues.
2. T35S Terminator - This terminator sequence is derived from the CaMV 35S promoter and can be used to terminate the expression of the beta-carotene hydroxylase gene in garlic.
3. NOS Polyadenylation Signal - This sequence is derived from the nopaline synthase (NOS) gene and is commonly used as a terminator sequence in plant genetic engineering. It can be used to terminate the expression of the alliinase, anthocyanin pathway, superoxide dismutase, and invertase genes in garlic.
**Vector & Delivery:** Vector: The Agrobacterium tumefaciens binary vector pCAMBIA 1300 will be an appropriate vector for delivering the desired genetic modifications into garlic plants. This vector has a broad host range, including Allium species, and allows the introduction of multiple genes at the same time. The pCAMBIA 1300 vector also carries a plant selectable marker (selection gene), which confers resistance to kanamycin or hygromycin. Selection gene is used to identify transformed cells and plants, which will facilitate the identification of garlic plants that have undergone the desired genetic modifications.
Delivery and Modification Method: Agrobacterium-mediated transformation will be the method of choice for delivering the desired genes into garlic plants. This method involves using a naturally occurring soil bacterium, Agrobacterium tumefaciens, as a delivery vehicle for transferring genes into plant cells. The bacterium is engineered to carry a modified plasmid that contains the desired genes under the control of the regulatory elements described above. The garlic plants are then infected with the engineered Agrobacterium, resulting in the transfer of the desired genes into the plant cells.
The infected cells are grown in selective media containing kanamycin or hygromycin, which kills cells that did not incorporate the selection gene. Transformed cells will grow and form calli, which can then be screened for the desired genetic modifications using PCR and other molecular biology methods. Transformed calli are then induced to regenerate into whole plants, which can be transferred to soil for further growth and evaluation.
Agrobacterium-mediated transformation is a widely used and effective method for delivering genes into plants, including garlic. Compared to other methods such as biolistics, it has a higher transformation efficiency and a lower risk of inducing random mutations or damage to the plant genome. Therefore, it is an appropriate method for achieving the desired genetic modifications in garlic plants efficiently and with high precision.
**Selection Marker:** If a selection marker is deemed needed for this project, we will use a kanamycin or hygromycin selectable marker carried on the pCAMBIA 1300 vector. This will facilitate the identification of transformed garlic plants that have incorporated the desired genetic modifications. The selectable marker will confer resistance to either kanamycin or hygromycin, allowing for the growth and selection of transformed cells on culture media containing the corresponding antibiotic. Functionally, this will make it easy to differentiate transformed cells from untransformed cells in culture, making it easier to screen and select transformed cells for further growth and analysis. Furthermore, the use of a selectable marker that is different from garlic's native antibiotic resistance will reduce the risk of horizontal gene transfer to opportunistic pathogenic bacteria, thus improving biosafety.
**Transformation Protocol:** Protocol for transforming garlic with selected genes:
Materials:
- Garlic bulbs for transformation
- Agrobacterium tumefaciens binary vector pCAMBIA 1300
- Antibiotics for selection (kanamycin or hygromycin)
- Murashige and Skoog (MS) medium
- Plant growth hormones (auxins and cytokinins)
- Sterilized instruments (scissors, forceps, scalpels)
- Sterile Petri dishes
- Culture flasks
- PCR primers for gene detection
- PCR machine
- DNA extraction kit
- Gel electrophoresis equipment
- Ethidium bromide stain
Steps:
1. Preparation of Agrobacterium tumefaciens with the desired plasmid:
- Grow Agrobacterium tumefaciens on a selective medium containing kanamycin or hygromycin overnight.
- Harvest the bacterial culture by centrifugation at 4000 rpm for 15 minutes.
- Resuspend the bacterial pellet in sterilized water or buffer.
2. Garlic bulbs surface sterilization and explant preparation:
- Clean the surface of the garlic bulb with 70% ethanol.
- Peel off the outermost layer of the garlic bulb to expose the cloves.
- Cut the cloves into small explants, approximately 5 mm in size.
- Place the explants in a sterile Petri dish.
3. Co-cultivation of garlic explants with Agrobacterium:
- Mix the Agrobacterium suspension with the garlic explants in a sterile environment, incubate at 25°C for 2-3 days, and enable co-cultivation.
- Carefully remove the explants from the suspension and place them onto a sterile MS medium with antibiotics (kanamycin or hygromycin).
4. Selection and regeneration of transformed cells:
- Culture the explants on a selection medium containing kanamycin or hygromycin, making sure to screen out non-transformed cells.
- Incubate the Petri dishes under controlled temperature and light conditions (25°C, 16h light/8h dark cycle).
- After 2-3 weeks, monitor the explants for the presence of calli, growing masses of undifferentiated cells.
- Identify calli which display the desired genetic modifications using PCR and electrophoresis of PCR products.
- Induce the regeneration of transformed cells by transferring them to a MS medium containing appropriate concentrations of auxins and cytokinins to encourage plant development.
5. Transplanting and acclimatization of transformed plants:
- After one month, transfer the plantlets to soil or a suitable substrate and grow them under controlled conditions (temperature, humidity, light).
- After acclimatization, transfer the plants to the field for further growth and evaluation of phenotypic traits.
6. Characterization of the transformed plants:
- Verify the genetic modifications in the regenerated plants using PCR and electrophoresis of DNA isolated from the leaves.
- Quantify the expression levels of the target genes through RT-PCR.
- Evaluate the phenotype of transformed garlic plants, examining traits such as allicin content, vitamin A content and antioxidant capacity.
- Compare phenotypes and genetic modifications between transformed and non-transformed groups to observe the impact of the desired genetic modifications on garlic's traits.
Note: Biosafety protocols must be followed to ensure that the transformed garlic plants do not present any environmental or health risks.
**Gene cassette**: The 5' to 3' list of elements in the multi-purpose cassette are as follows:
1. CaMV 35S Promoter - to drive the expression of the Beta-carotene hydroxylase gene for enhanced vitamin A production.
2. Beta-carotene hydroxylase gene - imported from other plants into garlic for enhanced production of vitamin A.
3. CaMV 35S Enhancer - to enhance the activity of the CaMV 35S promoter used to drive the beta-carotene hydroxylase gene.
4. T35S Terminator - derived from the CaMV 35S promoter and can be used to terminate the expression of the beta-carotene hydroxylase gene in garlic.
5. OinC Promoter - to drive the expression of the Alliinase gene for reducing garlic's pungent taste and odor.
6. 5' upstream sequence of the Alliinase gene - to enhance the expression of the Alliinase gene in garlic.
7. NOS Polyadenylation Signal - to terminate the expression of the Alliinase, Anthocyanin pathway genes, Superoxide dismutase, and Invertase genes in garlic.
8. Anthocyanin pathway genes - Dihydroflavonol reductase, Flavonoid 3',5'-hydroxylase, and Anthocyanidin synthase for enhanced garlic shelf-life and resistance to damage during storage.
9. PAL Enhancer - to enhance the expression of Phenylalanine ammonia-lyase and, subsequently, enhance the expression of the anthocyanin pathway genes in garlic.
10. Superoxide dismutase gene - to increase the level of ROS scavenging activity and enhance the antioxidant capacity of garlic.
11. Invertase gene - to enhance the sugar metabolism pathway and potentially increase the sugar content of garlic.
12. Nos Terminator - commonly used in plant genetic engineering and is known to function efficiently in various plant tissues to terminate the expression of the desired genes.
13. pCAMBIA 1300 Vector - a broad host range vector that allows the introduction of multiple genes at the same time and carries a plant selectable marker for identification of transformed garlic plants.
14. Agrobacterium-mediated transformation - an effective method for delivering genes into garlic plants with a higher transformation efficiency and a lower risk of inducing random mutations or damage to the plant genome.
15. Kanamycin or Hygromycin Selectable Marker - used to differentiate transformed cells from untransformed cells in culture and reduce the risk of horizontal gene transfer to opportunistic pathogenic bacteria, improving biosafety.
**Paper Abstract:** The main objective of this study is to use CRISPR Cas-9 gene editing technology and gene insertion techniques to modify garlic by introducing or knocking out genes associated with its nutritional and sensory properties. Specifically, we aim to reduce the pungency of garlic by knocking out or reducing the expression of the Alliinase gene, enhance its vitamin A production by inserting the Beta-carotene hydroxylase gene, improve its shelf-life by introducing anthocyanin pathway genes from blue corn maize, increase its antioxidant capacity by overexpressing the Superoxide dismutase gene, and enhance its sugar metabolism pathway by increasing the expression level of the Invertase gene. To achieve these objectives, we will use regulatory elements such as ideal promoter, enhancer, and terminator sequences that are active in garlic and other compatible plant species. We will deliver the desired genes into garlic plants using Agrobacterium-mediated transformation, a widely used and highly efficient method for delivering genes into plant cells.
The implications of this study are significant as it opens up new possibilities for modifying the nutritional and sensory properties of garlic. Specifically, the development of low-pungency, high vitamin A, and improved shelf-life garlic could have significant health benefits for consumers, especially those with vitamin A deficiency or who cannot consume highly pungent garlic due to health conditions. Furthermore, garlic could become a viable source of vitamin A, which is usually obtained from fruits and vegetables that are not always readily available or affordable in certain regions. The enhanced sugar content in garlic could also have implications for its use as a snack or culinary ingredient. Overall, the findings of this study could pave the way for further research on modifying other plant species to enhance their nutritional and sensory properties.
**Growth, Selection & Stabilization:** Growth and Selection Protocol:
1. Garlic plant growth conditions:
- Garlic plants should be grown in well-draining soil with a pH of 6.0-7.5 and adequate moisture.
- Temperature: 15-25°C
- Light: Garlic plants should be exposed to full sunlight for at least 6 hours per day.
- Nutrients: Garlic plants should be fertilized with a balanced fertilizer containing nitrogen, phosphorus, and potassium. Additional fertilization with micronutrients may be needed depending on soil conditions.
- Growth stage: Garlic plants should be grown until they reach the vegetative stage, approximately 6-8 weeks after planting.
2. Agrobacterium culture conditions:
- Agrobacterium tumefaciens strain carrying the desired plasmid should be grown in LB (Luria-Bertani) media supplemented with appropriate antibiotics (e.g. rifampicin) at 28°C with agitation.
3. Delivery of the desired genes into garlic plants:
- The garlic plants should be injured by cutting their basal plate with a scalpel to facilitate infection by the Agrobacterium.
- The Agrobacterium cells carrying the desired genes should be added to the garlic plant wound induced on the cutting plate.
- Infected garlic plants should be incubated in a humid environment for 48 hours to allow for Agrobacterium attachment and gene transfer.
4. Selection of transformed cells:
- Infected garlic plants should be transferred to a selection medium containing kanamycin (50 mg/L) or hygromycin (20 mg/L).
- Calli generated from the infected cells should be screened for the presence of the desired genetic modifications using PCR and sequencing. It is advisable to obtain a minimum of three regenerated plants for all gene-lines.
- Transformed calli are then induced to regenerate into whole plants, which can then be transferred to soil for further growth and evaluation.
5. Stabilization of the modified organisms:
- Transgenic garlic plants should be crossed with wild-type garlic plants to increase the genetic diversity and minimize the risk of unintended consequences.
- Stable lines of the modified garlic plants should be maintained in isolated fields, in separated greenhouses or by other physical means, preventing cross-pollination with non-GM garlic and minimizing gene flow.
- Regular monitoring for the presence of the introduced genes and their phenotypic consequences should be undertaken through regular use of PCR and phenotypic analysis under controlled conditions.
**Proliferation Method:** To proliferate the final transformants once stabilized, we will use tissue culture techniques to multiply the plants. Specifically, we will use a method known as micropropagation, which involves growing plant tissue on agar media in aseptic conditions. Individual garlic plants can be multiplied into large numbers of identical clones using this method.
To efficiently proliferate the desired genetic modifications in the resulting population, we will ensure that all the transformed plants display stable inheritance of the introduced genetic modifications. We will confirm this stability by conducting PCR analysis and other molecular biology methods at different stages of plant growth and development, including after callus formation, plant regeneration, and during the early stages of growth in soil.
To maintain the desired genetic modifications, we will avoid any cross-pollination or hybridization between the transformed garlic plants and other garlic plants in the vicinity. We will take measures such as growing the plants in a confined environment or using physical barriers to ensure that the transformed garlic plants do not cross with any other garlic plants. We will also isolate the plants that display the desired genetic modifications and grow them separately to avoid any unintended gene flow.
In summary, by using tissue culture techniques such as micropropagation, and taking measures to ensure genetic stability and prevent cross-pollination or hybridization, we can efficiently proliferate the desired genetic modifications in garlic plants and maintain these modifications in the resulting population for further study or commercialization.
**Conclusion:** In conclusion, the project aimed to modify garlic through genetic engineering techniques. The first protocol outlined the steps to introduce desired genes into garlic, using Agrobacterium-mediated transformation. After selection and regeneration of transformed cells, stable lines of the modified garlic plants were to be maintained to evaluate the phenotypic traits. The second protocol aimed to proliferate the final transformants once stabilized and maintain the desired genetic modifications to efficiently proliferate the desired genetic modifications. The third protocol suggested constructing a multi-purpose cassette with a list of elements that could modify garlic by reducing pungency, enhancing vitamin A production, improving shelf-life, increasing antioxidant capacity and sugar content. The significance of this study is significant, as the modifications could have considerable benefits for consumers and the agriculture industry. The study could pave the way for further research on developing other plant species to enhance their nutritional and sensory properties. However, biosafety protocols must be followed to ensure that these modified garlic plants do not pose any health or environmental hazards. Overall, the project has been successful in outlining protocols for garlic transformation, growth and selection, and proliferation, which could open new possibilities for modifying the nutritional and sensory properties of other plant species.