Unit- 9B

Biotechnology covering

Q1.What is Biotechnology?

Ans 1)

Biotechnology is technology that utilizes biological systems, living organisms or parts of this to develop or create different products.

Brewing and baking bread are examples of processes that fall within the concept of biotechnology (use of yeast (= living organism) to produce the desired product). Such traditional processes usually utilize the living organisms in their natural form (or further developed by breeding), while the more modern form of biotechnology will generally involve a more advanced modification of the biological system or organism.

With the development of genetic engineering in the 1970s, research in biotechnology (and other related areas such as medicine, biology etc.) developed rapidly because of the new possibility to make changes in the organisms' genetic material (DNA).

Today, biotechnology covers many different disciplines (eg. genetics, biochemistry, molecular biology, etc.). New technologies and products are developed every year within the areas of eg. medicine (development of new medicines and therapies), agriculture (development of genetically modified plants, biofuels, biological treatment) or industrial biotechnology (production of chemicals, paper, textiles and food).

Q2) What is Totipotency in biotechnology and totipotency in other factor?

A2)

Totipotency is the genetic potential of a plant cell to produce the entire plant. In other words, totipotency is the cell characteristic in which the potential for forming all the cell types in the adult organism is retained.

 Totipotency in Culture:

The basis of tissue culture is to grow large number of cells in a sterile controlled environ­ment. The cells are obtained from stem, root or other plant parts and are allowed to grow in culture medium containing mineral nutrients, vi­tamins and hormones to encourage cell division and growth. As a result, the cells in culture will produce an unorganised proliferative mass of cells which is known as callus tissue.

Totipotency in Plant Science:

The ultimate objective in plant protoplast, cell and tissue culture is the reconstruction of plants from the totipotent cell. Although the process of differentiation is still mysterious in general, the expression of totipotent cell in cul­ture has provided a lot of information’s.

Q3) How does manipulation and totipotency help in plants?

A3)

The ultimate objectives of any scientific investigation are twofold–to increase understanding, and to create opportunities for the application of the new knowledge, ostensibly in the service of man. The two objectives may not often be consciously connected in the minds of the investigators, but history and experience have demonstrated that new information always stimulates a drive towards practical application. This is already beginning to be true for plant cell biology through the directed manipulation of plant cells grown in culture.

Plant cell physiology and biochemistry is, at each moment in time, limited by the resolving power of current experimental techniques and by the availability of appropriate experimental plant material. Here we are concerned with the development of new experimental materials (cultured organs, tissues and cells), with culture systems being evolved for their exploitation and with recent experimental work which illustrates that these culture systems are adding a new dimension to studies in plant cell physiology.

The complexity of higher plants has inevitably led workers, concerned with investigating particular aspects of plant physiology, to the use of systems which are of reduced complexity. Hence the early and continuing use by plant physiologists of isolated plant organs (e.g. seedling roots, leaves), complex tissue systems (e.g. discs cut from leaves and storage organs) organ segments (e.g. segments of coleoptiles and hypocotyls) and isolated cell organelles. However, isolated organs and organ fragments are still systems of very considerable complexity and they are, from the beginning, systems of declining viability and favourable sites for colonization by microorganisms.

Changes in Growth and Metabolism of Plant Cells in Batch Culture-Cyto-differentiation

Plant cells in batch culture, i.e. cultures in a fixed volume of culture medium, increase in biomass by cell division and cell growth until a factor in the culture environment becomes limiting and sends them into a stationary phase. When such stationary phase cells are subcultured they pass in succession through a lag phase, a short-lived period of exponential growth, a period of declining relative growth rate and then again enter stationary phase (Fig. 15.1A). Traditionally such cultures are initiated by an inoculum establishing a relatively high initial cell density and only accomplish a very limited number of divisions before entering stationary phase. For example cell cultures of sycamore (Acer pseudoplatanus ) initiated at ca. 2 × 105 cells ml–1 will reach a final cell density of ca. 3 × 106 cells ml–1 corresponding to 4 successive doublings of the initial population.

Q4) How does manipulation and totipotency help in animals?

A4)

Molecular mechanisms defining Totipotency and cell differentiation in humans is a promising strategy in order to expand knowledge about reprogramming. Totipotency and the very first steps of cell differentiation can be studied well in early human embryos. Based on analysis of marker genes such as Oct-4 and β-HCG, blastomeres seem to differ in their potency and can be regarded as lineage-specific stem cells as early as the 4-cell stage. The allocation of these stem cells to specific fates might hereby follow a pattern reminiscent of animal and vegetal poles. On the opposite end of the developmental spectrum, differentiated human cells can be used as a means of studying nuclear reprogramming. Intact human 293T kidney cells and primary leukocytes were reprogrammed towards a more undifferentiated state by Xenopuslaevis egg extract. Molecular screens identified the chromatin-remodelling ATPase BRG1 as a factor required for this process. Based on these results, more efficient reprogramming protocols allowing for the generation of fully differentiated or undifferentiated human cells for clinical application may be developed.

Human cells provide another useful approach to study reprogramming. The establishment of protocols allowing for the targeted growth of reprogrammed cells from easily accessible and renewable sources might lead to future clinical application. Marker genes similar to ones used for human embryos can be employed to monitor reprogramming success. In addition, the large number of available cells for experimentation enables molecular screens, which allow for the identification of novel factors controlling transcription during reprogramming.

Q5) Method and uses of agriculture in biotechnology and crop modification technique?

A5)

Agricultural biotechnology, also known as agritech, is an area of agricultural science involving the use of scientific tools and techniques, including genetic engineering, molecular markers, molecular diagnostics, vaccines, and tissue culture, to modify living organisms: plants, animals, and microorganisms. Crop biotechnology is one aspect of agricultural biotechnology which has been greatly developed upon in recent times. Desired trait are exported from a particular species of Crop to an entirely different species. These transgene crops possess desirable characteristics in terms of flavor, colour of flowers, growth rate, size of harvested products and resistance to diseases and pests.

Traditional breeding

Traditional crossbreeding has been used for centuries to improve crop quality and quantity. Crossbreeding mates two sexually compatible species to create a new and special variety with the desired traits of the parents. For example, the honeycrisp apple exhibits a specific texture and flavor due to the crossbreeding of its parents. In traditional practices, pollen from one plant is placed on the female part of another, which leads to a hybrid that contains genetic information from both parent plants. Plant breeders select the plants with the traits they're looking to pass on and continue to breed those plants. Note that crossbreeding can only be utilized within the same or closely related species.

Mutagenesis

Mutations can occur randomly in the DNA of any organism. In order to create variety within crops, scientists can randomly induce mutations within plants. Mutagenesis uses radioactivity to induce random mutations in the hopes of stumbling upon the desired trait. Scientists can use mutating chemicals such as ethyl methanesulfonate, or radioactivity to create random mutations within the DNA. Atomic gardens are used to mutate crops. A radioactive core is located in the center of a circular garden and raised out of the ground to radiate the surrounding crops, generating mutations within a certain radius. Mutagenesis through radiation was the process used to produce ruby red grapefruits.

Polyploidy

Polyploidy can be induced to modify the number of chromosomes in a crop in order to influence its fertility or size. Usually, organisms have two sets of chromosomes, otherwise known as a diploidy. However, either naturally or through the use of chemicals, that number of chromosomes can change, resulting in fertility changes or size modification within the crop. Seedless watermelons are created in this manner; a 4-set chromosome watermelon is crossed with a 2-set chromosome watermelon to create a sterile (seedless) watermelon with three sets of chromosomes.

Protoplast fusion

Protoplast fusion is the joining of cells or cell components to transfer traits between species. For example, the trait of male sterility is transferred from radishes to red cabbages by protoplast fusion. This male sterility helps plant breeders make hybrid crops.[4]

RNA interference

RNA interference (RNAIi) is the process in which a cell's RNA to protein mechanism is turned down or off in order to suppress genes. This method of genetic modification works by interfering with messenger RNA to stop the synthesis of proteins, effectively silencing a gene.

Transgenics

Transgenics involves the insertion of one piece of DNA into another organism's DNA in order to introduce new genes into the original organism. This addition of genes into an organism's genetic material creates a new variety with desired traits. The DNA must be prepared and packaged in a test tube and then inserted into the new organism. New genetic information can be inserted with biolistics. An example of transgenics is the rainbow papaya, which is modified with a gene that gives it resistance to the papaya ringspot virus.

Genome editing

Genome editing is the use of an enzyme system to modify the DNA directly within the cell. Genome editing is used to develop herbicide resistant canola to help farmers control weed.

Q6) How to improve nutritional content in agriculture biotechnology?

A6)

Agricultural biotechnology has been used to improve the nutritional content of a variety of crops in an effort to meet the needs of an increasing population. Genetic engineering can produce crops with a higher concentration of vitamins. For example, golden rice contains three genes that allow plants to produce compounds that are converted to vitamin A in the human body. This nutritionally improved rice is designed to combat the world's leading cause of blindness—vitamin A deficiency. Similarly, the Banana 21 project has worked to improve the nutrition in bananas to combat micronutrient deficiencies in Uganda. By genetically modifying bananas to contain vitamin A and iron, Banana 21 has helped foster a solution to micronutrient deficiencies through the vessel of a staple food and major starch source in Africa. Additionally, crops can be engineered to reduce toxicity or to produce varieties with removed allergens.

Genes and traits of interest for crop

Agronomic trait

Insect resistance

One highly sought after trait is insect resistance. This trait increases a crop's resistance to pests and allows for a higher yield. An example of this trait are crops that are genetically engineered to make insecticidal proteins originally discovered in (Bacillus thuringiensis). Bacillus thuringiensis is a bacteria that produces insect repelling proteins that are non-harmful to humans. The genes responsible for this insect resistance have been isolated and introduced into many crops. Bt corn and cotton are now commonplace, and cowpeas, sunflower, soybeans, tomatoes, tobacco, walnut, sugar cane, and rice are all being studied in relation to Bt.

Herbicide tolerance Weeds have proven to be an issue for farmers for thousands of years; they compete for soil nutrients, water, and sunlight and prove deadly to crops. Biotechnology has offered a solution in the form of herbicide tolerance. Chemical herbicides are sprayed directly on plants in order to kill weeds and therefore competition, and herbicide resistant crops have to the opportunity to flourish.

Disease resistance

Often, crops are afflicted by disease spread through insects (like aphids). Spreading disease among crop plants is incredibly difficult to control and was previously only managed by completely removing the affected crop. The field of agricultural biotechnology offers a solution through genetically engineering virus resistance. Developing GE disease-resistant crops now include cassava, maize, and sweet potato.

Temperature tolerance

Agricultural biotechnology can also provide a solution for plants in extreme temperature conditions. In order to maximize yield and prevent crop death, genes can be engineered that help to regulate cold and heat tolerance. For example, papaya trees have been genetically modified in order to be more tolerant of hot and cold conditions.[6] Other traits include water use efficiency, nitrogen use efficiency and salt tolere.

Quality traits

Quality traits include increased nutritional or dietary value, improved food processing and storage, or the elimination of toxins and allergens in crop plants.

Q7) Describe how to work biotechnology in health and medicine?

A7)

Healthcare biotechnology refers to a medicinal or diagnostic product or a vaccine that consists of, or has been produced in, living organisms and may be manufactured via recombinant technology (recombinant DNA is a form of DNA that does not exist naturally. It is created by combining DNA sequences that would not normally occur together). This technology has a tremendous impact on meeting the needs of patients and their families as it not only encompasses medicines and diagnostics that are manufactured using a biotechnological process, but also gene and cell therapies and tissue engineered products. Biotechnology offers patients a variety of new solutions such as: Unique, targeted and personalized therapeutic and diagnostic solutions for particular diseases or illnesses, An unlimited amount of potentially safer products, Superior therapeutic and diagnostic approaches, Higher clinical effectiveness because of the biological basis of the disease being known, Development of vaccines for immunity, Treatment of diseases, Cultured Stem Cells and Bone Marrow Transplantation, Skin related ailments and use of cultured cell, Genetic Counseling, Forensic Medicine, Gene Probes, Genetic Fingerprinting, Karyotyping.

Q8) Recombinant of DNA Technology technique and application?

A8)

Recombinant DNA, molecules of DNA from two different species that are inserted into a host organism to produce new genetic combinations that is of value to science, medicine, agriculture, and industry. Since the focus of all genetics is the gene, the fundamental goal of laboratory geneticists is to isolate, characterize, and manipulate genes. Although it is relatively easy to isolate a sample of DNA from a collection of cells, finding a specific gene within this DNA sample can be compared to finding a needle in a haystack. Consider the fact that each human cell contains approximately 2 metres (6 feet) of DNA. Therefore, a small tissue sample will contain many kilometres of DNA. However, recombinant DNA technology has made it possible to isolate one gene or any other segment of DNA, enabling researchers to determine its nucleotide sequence, study its transcripts, mutate it in highly specific ways, and reinsert the modified sequence into a living organism.

In biology a clone is a group of individual cells or organisms descended from one progenitor. This means that the members of a clone are genetically identical, because cell replication produces identical daughter cells each time. The use of the word clone has been extended to recombinant DNA technology, which has provided scientists with the ability to produce many copies of a single fragment of DNA, such as a gene, creating identical copies that constitute a DNA clone. In practice the procedure is carried out by inserting a DNA fragment into a small DNA molecule and then allowing this molecule to replicate inside a simple living cell such as a bacterium. The small replicating molecule is called a DNA vector (carrier). The most commonly used vectors are plasmids (circular DNA molecules that originated from bacteria), viruses, and yeast cells. Plasmids are not a part of the main cellular genome, but they can carry genes that provide the host cell with useful properties, such as drug resistance, mating ability, and toxin production. They are small enough to be conveniently manipulated experimentally, and, furthermore, they will carry extra DNA that is spliced into them.

Q9) What is DNA Cloning and how to create clone?

A9)

Clone is a group of individual cells or organisms descended from one progenitor. This means that the members of a clone are genetically identical, because cell replication produces identical daughter cells each time. The use of the word clone has been extended to recombinant DNA technology, which has provided scientists with the ability to produce many copies of a single fragment of DNA, such as a gene, creating identical copies that constitute a DNA clone. In practice the procedure is carried out by inserting a DNA fragment into a small DNA molecule and then allowing this molecule to replicate inside a simple living cell such as a bacterium. The small replicating molecule is called a DNA vector (carrier). The most commonly used vectors are plasmids (circular DNA molecules that originated from bacteria), viruses, and yeast cells. Plasmids are not a part of the main cellular genome, but they can carry genes that provide the host cell with useful properties, such as drug resistance, mating ability, and toxin production. They are small enough to be conveniently manipulated experimentally, and, furthermore, they will carry extra DNA that is spliced into them.

Creating the clone

The steps in cloning are as follows. DNA is extracted from the organism under study and is cut into small fragments of a size suitable for cloning. Most often this is achieved by cleaving the DNA with a restriction enzyme. Restriction enzymes are extracted from several different species and strains of bacteria, in which they act as defense mechanisms against viruses. They can be thought of as “molecular scissors,” cutting the DNA at specific target sequences. The most useful restriction enzymes make staggered cuts; that is, they leave a single-stranded overhang at the site of cleavage. These overhangs are very useful in cloning because the unpaired nucleotides will pair with other overhangs made using the same restriction enzyme. So, if the donor DNA and the vector DNA are both cut with the same enzyme, there is a strong possibility that the donor fragments and the cut vector will splice together because of the complementary overhangs. The resulting molecule is called recombinant DNA. It is recombinant in the sense that it is composed of DNA from two different sources. Thus, it is a type of DNA that would be impossible naturally and is an artifact created by DNA technology.

The next step in the cloning process is to cut the vector with the same restriction enzyme used to cut the donor DNA. Vectors have target sites for many different restriction enzymes, but the most convenient ones are those that occur only once in the vector molecule. This is because the restriction enzyme then merely opens up the vector ring, creating a space for the insertion of the donor DNA segment. Cut vector DNA and donor DNA are mixed in a test tube, and the complementary ends of both types of DNA unite randomly. Of course, several types of unions are possible: donor fragment to donor fragment, vector fragment to vector fragment, and, most important, vector fragment to donor fragment, which can be selected for. Recombinant DNA associations form spontaneously in the above manner, but these associations are not stable because, although the ends are paired, the sugar-phosphate backbone of the DNA has not been sealed. This is accomplished by the application of an enzyme called DNA ligase, which seals the two segments, forming a continuous and stable double helix.

The mixture should now contain a population of vectors each containing a different donor insert. This solution is mixed with live bacterial cells that have been specially treated to make their cells more permeable to DNA. Recombinant molecules enter living cells in a process called transformation. Usually, only a single recombinant molecule will enter any individual bacterial cell. Once inside, the recombinant DNA molecule replicates like any other plasmid DNA molecule, and many copies are subsequently produced. Furthermore, when the bacterial cell divides, all of the daughter cells receive the recombinant plasmid, which again replicates in each daughter cell.

Q10) What is the role recombinant DNA technology for improve life?

A10)

The recombinant DNA technology was just an imagination that desirable characteristics can be improved in the living bodies by controlling the expressions of target genes. However, in recent era, this field has demonstrated unique impacts in bringing advancement in human life. By virtue of this technology, crucial proteins required for health problems and dietary purposes can be produced safely, affordably, and sufficiently. This technology has multidisciplinary applications and potential to deal with important aspects of life, for instance, improving health, enhancing food resources, and resistance to divergent adverse environmental effects. Particularly in agriculture, the genetically modified plants have augmented resistance to harmful agents, enhanced product yield, and shown increased adaptability for better survival. Moreover, recombinant pharmaceuticals are now being used confidently and rapidly attaining commercial approvals. Techniques of recombinant DNA technology, gene therapy, and genetic modifications are also widely used for the purpose of bioremediation and treating serious diseases. Due to tremendous advancement and broad range of application in the field of recombinant DNA technology, this review article mainly focuses on its importance and the possible applications in daily life.

Recombinant DNA Technology

Recombinant DNA technology comprises altering genetic material outside an organism to obtain enhanced and desired characteristics in living organisms or as their products. This technology involves the insertion of DNA fragments from a variety of sources, having a desirable gene sequence via appropriate vector. Manipulation in organism's genome is carried out either through the introduction of one or several new genes and regulatory elements or by decreasing or blocking the expression of endogenous genes through recombining genes and elements. Enzymatic cleavage is applied to obtain different DNA fragments using restriction endo-nucleases for specific target sequence DNA sites followed by DNA ligase activity to join the fragments to fix the desired gene in vector. The vector is then introduced into a host organism, which is grown to produce multiple copies of the incorporated DNA fragment in culture, and finally clones containing a relevant DNA fragment are selected and harvested. The first recombinant DNA (rDNA) molecules were generated in 1973 by Paul Berg, Herbert Boyer, Annie Chang, and Stanley Cohen of Stanford University and University of California San Francisco. In 1975, during “The Asilomar Conference” regulation and safe use of rDNA technology was discussed. Paradoxically to the view of scientists at the time of Asilomar, the recombinant DNA methods to foster agriculture and drug developments took longer than anticipated because of unexpected difficulties and barriers to achieve the satisfactory results. However, since the mid-1980s, the number of products like hormones, vaccines, therapeutic agents, and diagnostic tools has been developed continually to improve health.