Formal Lab Report Plant Bio Paper
Advances in the technology have been due to development of a range of Crematorium-mediated and direct DNA delivery techniques, along with appropriate tissue culture techniques or regenerating whole plants from plant cells or tissues in a large number of species Crematorium-mediated genetic transformation is the dominant technology used for the production of genetically modified transgenic plants. Crematorium tenancies is a naturally occurring gram negative bacterium which causes crown gall disease in over 140 species of idiotic plants.
The strains used for transforming Rhapsodies contain T-DNA in the It plasmid which are then introduced into the plant cell. This It plasmid releases the T-DNA once in the plant cell which gets incorporated into a semi-random location in the plant gnome. Once incorporated, T-DNA induces the plant to produce hormones such as again and cytokine which are required for Crematorium nutrition. The gene of interest and plant selection marker are maintained within the T-DNA region of the T-DNA binary vector.
The IVR protein genes are on a separate replication called It helper plasmid. This helper plasmid mediates the T-DNA processing from the binary vector and T-DNA transfer from the bacterium to the host cell Extensive research aimed at understanding and improving the molecular machinery of Crematorium responsible for the generation and transport of the bacterial DNA into the host cell has resulted in the establishment of many recombinant Crematorium strains, plasmids and technologies currently used for the successful transformation of numerous plant species.
Somatic embryos have been successfully used as a target tissue for transformation and regeneration of transgenic plants. Somatic embryos, initiated originally from developing zygotic embryos, proliferate numerous secondary embryos from single cells in the epidermal layer. These single cells in intact somatic embryos are susceptible to transformation by genetically engineered Crematorium tenancies ND provide a means to regenerate transgenic plants. The genetic material that is introduced is called T DNA (transferred DNA) which is located on a It plasmid.
A It plasmid is a circular piece of DNA found in almost all bacteria. This gene transfer system has been made more efficient using, a) vector plasmids containing two marker genes encoding џ-cloudiness (GUS) and macroeconomics phosphorescence’s and B) a more virulent strain of Crematorium. This system should be applicable to any crop that undergoes repetitive embryologists from single Crematorium-susceptible cells. Crematorium tenancies is a widespread naturally occurring soil bacterium that causes crown gall, and has the ability to introduce new genetic material into the plant cell.
This natural ability to alter the plant’s genetic makeup was the foundation of plant transformation using Crematorium. Currently, Crematorium-mediated transformation is the most commonly used method for plant genetic engineering because of relatively high efficiency. Initially it was believed that this Crematorium only infects dicotyledonous plants, but it was later established that it can also be used for transformation of monocotyledonous plants such as rice.
During transformation, several components of the It plasmid enable effective transfer of the genes of interest into the plant cells. These include: T-DNA border sequences, which demarcate the DNA segment (T-DNA) to be transferred into the plant genome. IVR genes (virulence genes), which are required for transferring the T-DNA region to the plant but are not themselves transferred, and modified T-DNA region where the genes that cause crown gall formation are removed and replaced with the genes of interest. In this experiment Rhapsodies thaliana was transformed with
Crematorium tenancies which carried the vector. The vector (pacifically) transported by Crematorium contained GUS reporter gene driven by Napkin promoter which tested for the activity of our gene of interest. Materials and methods: Materials: Plant leaves (Rhapsodies thaliana and tomato). Scalpels Forceps Empty sterile plant Petri dishes Beakers Sterile filter paper disks Method: Crematorium tenancies strain GIVING containing amp (It-helper) plasmid and pacifically -Napkin (T-DNA) plasmid was used for plant transformation. Comparable-Napkin plasmid was constructed by cloning Brassier ARPA seed pacific Napkin promoter to control expression of GUS (beta-cloudiness) reporter gene (Figure 1). For plant transformation Crematorium was grown in LB media containing 50 g/ml of Jamaican for 24 hrs at 30 C with shaking at 200 RPM. Figure 1: pacifically used as vector for Rhapsodies transformation. Napkin promoter controls the expression of reporter gene guys week 2 The soil to be used for the planting seeds was saturated with water. The seeds were mixed with 1 ml of 0. 1% agrees and vortexes.
The entire contents of the seeds were sewed in the pot with the help of a pipette. The plants were then grown at ICC in light for 16 hrs. In light and 8 hrs. In the dark for 4-seeks until the leaves grew. Week 8 For transformation crematorium whole leaves from the plant were removed and were washed with warm water. Then the leaves were dipped in 70% ethanol in a sterile Petri dish and was rinsed with water. Leaves were sterile in 10% chlorine bleach for 5 min. They were then washed three times and were transferred to sterile empty Petri dishes =.
In a clean room sterile scalpels were used to cut leaves into square sections of about 1- 1. 5 CM square. While terrorizing plant material Crematorium was prepared for transformation. Crematorium culture was taken and was grown in LB+sank media. Following that 40 ml Agro culture was poured into sterile ml Falcon tube and was spun for 10 min at Max speed in the beach top centrifuge. After centrifuging supernatant was removed by pouring into a beaker or flask – this was to be autoclaves. Ml sterile lax MS salt solution was then added to the cells in the falcon tube.
Bacteria was responded by gently shaking the tube. In a clean room Crematorium culture was added to Petri dish containing plant leaves ND were agitated for 3-5 min by gently swirling. The leaves were then blotted. After blotting leaf disks were placed on Co-cultivation (SAC) medium and plates were incubated for 1-2 days in the dark or dim light. Successful germination and plant growth requires appropriate soil moisture, nutrient levels, light intensity, humidity, and temperature. If any of these are compromised, Rhapsodies will respond by flowering early and dying prematurely.
Washing off Crematorium and plating on antibiotic -shooting media: Plant leaves from co-cultivation media were removed and were washed in sterile water containing 500 MGM/L municipal 3-4 times in sterile Petri dishes. Then the leaves were washed in MS media containing 500 MGM/L inclining were blotted gently in Petri dish with filter paper. Sterile forceps were used to transfer leaves to shoot selective media (ASS) containing 1 00 MGM/L Jamaican and 100 MGM/L suffocative (or 500 MGM/L municipal (or better corbelling).
Week 11 Growth of shoots was recorded on the shoot selective media and then the leaves were transferred to root selective media and growth was recorded. Results: In this experiment Rhapsodies thaliana was transformed with Crematorium tenancies which carried the vector. The vector transported by Crematorium contained GUS reporter gene driven by Napkin promoter which tested for the activity of our gene of interest. The growth of the plants over the span of 11 weeks is described below. Week 2: Seeds were planted Week 5: Plants started to grow and produce leaves Week 8: 100 leaves were taken from the plant and were transferred Week 1 1: 11. 3596 % of transformation was seen in tomato leaves and 22. 38806 % of transformation was seen in Rhapsodies leaves. The leaves were not transferred to the root selective media because the leaves were contaminated in shoot selective media. Table 1: Growth of Rhapsodies and tomato in shoot and root growth media plates of 10 groups, observed during week 11 Group Tomato leaves Tomato shoots Rhapsodies leaves Rhapsodies shoots 7 6 2 11 3 10 0 4 20 5 Total 89 67 15 Discussion: The purpose of the lab was to obtain transgenic plants by leaf disk co- cultivation with crematorium tenancies containing foreign genes.
Firstly, to make crematorium less harmful to plants Crematorium virulence genes were removed from T-DNA and replaced with plant transformation selection marker and gene of interest ( antibiotic resistant gene). Following that the Rhapsodies eaves obtained were wounded ( cut into smaller pieces) and incubated with crematorium for two days. During this binding of Crematorium to a plant cell occurred (up to 200/cell can attach) and transfer of DNA to the plant cell took place (multiple T-Dana can be transferred).
Then after two days the leaves were removed and were washed with antibiotic and water. The leaves were washed with antibiotic to make sure it does not wash off the and stays in the leaves. Following that they were placed on antibiotic shooting medium containing 1 ml Jamaican stock (100 MGM/ml), 1 ml suffocative stock (100 MGM/ml) and 1 ml rebelling or municipal stock (1 MGM/ml), for a week. The leaves were grown on this particular media composition to confirm for the successful transfer of anti- biotic resistant gene in them.
Shoot which were observed a week later confirmed that our transformation was successful. After a week we were supposed to transfer the leaves to the rooting media. Shooting media helps in shoot formation of the explants and rooting media helps in the root formation of the explants. And roots help take up the nutrients which are used by the plant for its further growth. Unfortunately the leaves could not be transferred to the rooting Edie due to their contamination.
Our experiment was successful because a lot of shoots were growing from the explants. But due to the contamination of the explants we failed to transfer the shoots to the rooting media and observe for the growth of any roots. The contamination could have occurred due to a human error while transferring the explants to the shooting media. The plates of all the groups were contaminated shows that this could be due to the use of non-sterilized equipment. To further confirm the successful transformation Gus staining and DNA analysis is done.
For future experiments care should be taken hill transferring the explants from one media to the other. Making sure that equipment’s used are sterilized. Also. For future experiments other factors such as resistance to pesticides could also performed. After performing this lab it can be concluded that antibiotic resistance can be introduce into plants by using crematorium mediated transformation. Rhapsodies thaliana stenotype Columbia was used for Crematorium-mediated transformation by somatic method.
Crematorium-mediated Plant Transformation Process The Crematorium-mediated transformation process involves a number of steps: a) isolation of the genes of interest from the source organism; (b) development of a functional transgenic construct including the gene of interest; promoters to drive expression; code modification, if needed to increase successful protein production; and marker genes to facilitate tracking of the introduced genes in the host plant; (c) insertion of the transgender into the It-plasmid; (d) introduction of the T-DNA-containing-passim into Crematorium; (e) mixture of the transformed Crematorium with plant cells to allow transfer of T-DNA into plant chromosome; (f) regeneration of the transformed cells into genetically modified GM) plants; and (g) testing for trait performance or transgender expression at lab, greenhouse and field level.
Figure 1 illustrates Crematorium-mediated plant transformation.. The overall advantages of using Crematorium-mediated transformation over other transformation methods are: reduction in transgender copy number, and intact and stable integration of the transgender (newly introduced gene) into the plant genome In addition, parallel developments in molecular biology have greatly extended the range of investigations to which plant transformation technology can be applied. Research in plant transformation is concentrating now not so much on the introduction of DNA into plant cells, but rather more on the problems associated with stable integration and reliable expression of the DNA once it has been integrated.