The Importance of Biotechnology in Today’s Time

Biotechnology is the third wave in biological science and represents such an interface of basic and applied sciences, where gradual and subtle transformation of science into technology can be witnessed. Biotechnology is defined as the application of scientific and engineering principals to the processing of material by biological agents to provide goods and services. Biotechnology comprises a number of technologies based upon increasing understanding of biology at the cellular and molecular level.

The Bible already provides numerous examples of biotechnology. Namely, it deals with the conversion of grapes to wine, of dough to bread and of milk to cheese. The oldest biotechnological processes are found in microbial fermentations, as born out by the Babylonian tablet dated circa 6000 B.C., explaining the preparation of beer. The Sumerians were able to brew as many as twenty types of beer in the third millennium B.C. In about 4000 B.C. leavened bread was produced with the aid of yeast. During Vedic period (5000-7000 B.C.) Aryans had been performing daily Agnihotra or Yajna. In Ayurved, production of ‘Asava’ and ‘Arista’ using different substrates and flowers of mahua (Madhuca indica) or dhataki (Wodfordiafructicosa) has been well characterized till today since Vedic period. One of the materials used in Yajna is animal fat (i.e. ghee) which is fermented product of milk. The term ‘biotechnology’ was described in a Bulletin of the Bureau of Biotechnology published in July, 1920 from the office of the same name in Leeds in Yorkshire. The articles in this bulletin described the varied roles of microbes in leather industry to pest control.

There are numerous sub-fields of biotechnology. They are:

1. Red biotechnology is biotechnology applied to medical processes. Some examples are the designing of organisms to produce antibiotics, and the engineering of genetic cures to cure diseases through genomic manipulation.
2. White biotechnology, also known as grey biotechnology, is biotechnology applied to industrial processes. An example is the designing of an organism to produce a useful chemical. White biotechnology tends to consume less in resources than traditional processes when used to produce industrial goods.
3. Green biotechnology is biotechnology applied to agricultural processes. An example is the designing of an organism to grow under specific environmental conditions or in the presence (or absence) of certain agricultural chemicals. One hope is that green biotechnology might produce more environmentally friendly solutions than traditional industrial agriculture. An example of this is the engineering of a plant to express a pesticide, thereby eliminating the need for external application of pesticides. Whether or not green biotechnology products such as this are ultimately more environmentally friendly is a topic of considerable debate.
4. The term blue biotechnology has also been used to describe the marine and aquatic applications of biotechnology, but its use is relatively rare.

Broadly biotechnology can be divided into two major branches:

1. Non-gene biotechnology– deals with whole cell, tissues or even individual organisms
2. Gene biotechnology– involves gene manipulation, cloning, etc.

Non-gene biotechnology is a more popular practice, and plant tissue culture, hybrid seed production, microbial fermentation, production of hybridoma antibodies or immunochemicals are wide spread biotechnology practices.

For centuries humans have used microorganisms to produce foods and drinks without understanding the microbial processes underlying their production. In recent years the understanding of the biosynthetic pathways and regulatory control mechanisms used by microorganisms for production of several metabolites has been increased by developing the knowledge of biochemistry of industrially important organisms. Notable biotechnologies for food processing include fermentation technology, enzyme technology and monoclonal antibody technology. Beneficial microbes participate in fermentation processes, producing many useful metabolites such as enzymes, organic acids, solvents, vitamins, amino acids, antibiotics, growth regulators, flavors and nutritious foods. Some leading food bioprocessing technologies are dairy processing, alcohol and beverage processing. Production of alcoholic beverages include: wine, beer, whiskey, rum, shake, etc. utilizing microorganisms like Clostridium acetobutylicum, Lecuonostoc mesenteroides, Aspergillus oryzae,Saccharomyces cerevisiae, Rizopus sp., Mucor sp., etc. Biotechnologically produced organic acids like citric acid, acetic acid, gluconic acid, D-Lactic acid, fumaric acid, etc. also has very high market value.

The application of biotechnology can result in (a) new ways of producing existing products with the use of new inputs, and (b) new ways of producing new products. Examples of the former include the production of gasoline from ethanol which in turn is produced from sugar; the production of insulin using recombinant DNA technology; the production of hepatitis B vaccine using recombinant DNA technology and the extraction of copper using mineral leaching bacteria. The alternative inputs are oil for gasoline, porcine prancreases for insulin, human blood for hepatitis vaccine, and the conventional mining techniques for copper. Examples of the latter include possible medicinal substances which are produced in minute quantity in the human body and which cannot be synthesized such as insulin, interleukin or Tissue Plasminogen Activator (TPA).

A wide variety of microorganisms are now being employed as tools in biotechnology to produce useful products or services. Raw materials can be converted to useful finished products both by ordinary chemical processes and by biological means. Generally, the costs of chemical conversion are quite high as the reactions require high temperature or pressure. In contrast, biological alternatives, using microbes or cultured animal or plant cells, operate at physiologically normal conditions of temperature, pressure, pH, etc. During the next few decades biotechnology would have overtaken chemical technology, and many such chemicals which are today produced chemically would be made through biotechnology.

Enzyme technology is an area of considerable current interest and development. Enzymes are biological catalysts and have been used for many years as isolated agents particularly in food e.g. rennin, papain and invertase. These enzymes have increasingly replaced plants and animal enzymes; thus amylase from Bacillus and Aspergillus have substituted those of malted wheat and barley in brewing, baking and biscuit-making and also in the textile industry, etc. Today, enzyme technologies have four distinct areas of application: in cosmetics, therapy, the food and feed industry, and for diagnostic purposes. One very important recent application is the production of foodstuffs from non-traditional raw materials: for instance, the development of the sweetener, high fructose corn syrup (HFCS), also called isoglucose. Another recent application is the use of phytase in animal feed.

Nowadays, interest in the traditional fermentation technology for food processing has greatly increased because of emphasis placed upon plant materials as human foods. Single-cell protein (SCP) is term generally accepted to mean the microbial cells (algae, bacteria, actinomycetes and fungi) grown and harvested for animal or human food. During World War II, when there were shortages in proteins and vitamins in the diet, the Germans produced yeasts and a mold (Geotrichum candidum) in some quantity for food. Research on SCP has been stimulated by a concern over the eventual food crisis or food shortages that will occur if the world’s population is not controlled. Many scientists believe that the use of microbial fermentations and the development of an industry to produce and supply SCP are possible solutions to meet a shortage of protein if and when the amount of protein produced or obtained by agriculture and fishing becomes insufficient.

The roots of molecular biology were established only after the British biophysicist Francis Crick and the American biochemist James Watson, in 1953, proposed the structure of DNA (deoxyribonucleic acid) molecule which is well known as the chemical bearer of genetic information of most of the organisms. We really began understanding and utilizing molecular biotechnology (or gene biotechnology) only after recombinant DNA technology was developed in 1970’s. Daniel Nathans (in 1971) of John Hopkins University utilized the restriction enzyme to split DNA of monkey tumor virus, Simian Virus (SV40). Recombinant DNA technology, often referred to as genetic engineering or gene manipulation, involves extraction of a particular gene of interest form one organism and then insertion of the gene into other organisms. Genetic manipulation may be defined as the extracellular (i.e. in vitro) creation of new forms of arrangements of DNA in such a way as to allow the incorporation or continued propagation of altered genetic condition in nature. Among the first scientist to attempt genetic manipulation was Paul Berg of Stanford University who in 1971 along with his co-workers opened the DNA molecule of SV40 and spliced it into a bacterial chromosome and constructed the first recombinant DNA molecule.

The genetic engineering techniques are useful tools for genetic research. They can help to gain in the structure, function and regulation of genes. They also help to prepare the physical maps of viral genome. Maps of several viruses have been made available like SV40, Polyoma virus and adenovirus. Another goal in genetic engineering is to design super bug which can degrade most of the major hydrocarbon components of petroleum. The different strains of Pseudomonas putida contain a plasmid which has genes coding for enzymes that digest a single family of hydrocarbons. By crossing the various strains of this bacterium, a super bug has been created. The multiplasmid bacterium is able to grow on a diet of crude oil. The super bug has potential for clearing up oil spills.

Biotechnology is widely used in pharmacy to create more efficient and less expensive drugs. Recombinant DNA technology is used for production of specific enzymes, which enhance the rate of production of particular range of antibodies in the organism. The hormones such as somatostatin, insulin and the human growth hormone can be synthesized easily and cheaply. The first human hormone to be synthesized by genetic engineering was somatostatin. Somatostatin is brain hormone originating from hypothalamus. It acts to inhabit the release of human growth hormone and insulin is related to treatment of diabetes, pancreatis and few other conditions. Genetech, a California based company, has produced human growth hormone (hGH) from genetically engineered bacteria. Human insulin or humulin is the first genetically engineered pharmaceutical product, developed by Eli Lilly and company in 1982. Bovine Somatotropin (BST) is produced for a large quantity of milk production in cows. Antibiotics are chemical substances produced by several microorganisms. Recombinant DNA technology has helped in increased production of antibiotics; for example, the rate of penicillin produced at present is about 150,000 unit/ml against about 10 unit/ml in 1950s. Antibiotics produced using such technology have very specific effects and cause fewer side effects. Currently, scientists are working on vaccines for fatal illnesses such as AIDS, hepatitis, malaria, flu, and even some forms of cancer. Interferon, an anti-viral protein, is prepared from the mammalian cells by recombinant DNA technology. By cloning cDNA to genes for human interferon, it has been found that there are large number of interferon differing in amino acid sequences and properties. A large number of interferon is prepared in yeast cells by fermentation process. Shrof expects that in the near future vaccines will come in more convenient ways “some will come in the form of mouthwash; others will be swallowed in time-release capsules, avoiding the need for boosters.” (Shrof 57)

One of the best known applications of genetic engineering is that of the creation of genetically modified organisms (GMOs). There are potentially momentous biotechnological applications of GM, for example oral vaccines produced naturally in fruit, at very low cost. This represents, however, a spread of genetic modification to medical purposes and opens an ethical door to other uses of the technology to directly modify human genomes. A genetically modified food is a food product derived in whole or part from a genetically modified organism (GMO) such as a crop plant, animal or microbe such as yeast. Genetically modified foods have been available since the 1990s. The principal ingredients of GM foods currently available are derived from genetically modified soybean, maize and canola. Between 1996 and 2001, the total surface area of land cultivated with GMOs had increased by a factor of 30, from 17,000 km² (4.2 million acres) to 520,000 km² (128 million acres). The value for 2002 was 145 million acres (587,000 km²) and for 2003 was 167 million acres (676,000 km²). (Internet 6) Future applications of GMOs include bananas that produce human vaccines against infectious diseases such as Hepatitis B, fish that mature more quickly, fruit and nut trees that yield years earlier, and plants that produce new plastics with unique properties. Now scientists have transformed Tobacco Mosaic Virus (TMV) to infect host plants and produce immunizing proteins rather than debilitating leaf shrivel, turning greenhouse tobacco into a biofactory for plague vaccine.

Genetic diseases could be treated through the use of genetic engineering. Defective genes in an organism cause genetic disorders. If a defective gene could be identified and located in a particular group of cells – it could be replaced with a functional one. The transgenic cells are then planted into the organism, resulting in a cure of the disorder. Cloning is a relatively new sector of biotechnology, but it promises answers to very important problems related to surgery. Tissues and organs could be cloned for surgical purposes. If scientists could isolate stem cells and then direct their development, they would be able to create any kind of a tissue, organ or even a whole part of a body.

Another revolutionizing tool of biotechnology is DNA fingerprinting. DNA fingerprints are useful in several applications of human health care research, as well as in the justice system. DNA fingerprinting is used to diagnose inherited disorders in both prenatal and newborn babies in hospitals around the world. These disorders may include cystic fibrosis, hemophilia, Huntington’s disease, familial Alzheimer’s, sickle cell anemia, thalassemia, and many others. Early detection of such disorders enables the medical staff to prepare themselves and the parents for proper treatment of the child. In some programs, genetic counselors use DNA fingerprint information to help prospective parents understand the risk of having an affected child. DNA fingerprint information can also help in developing cures for inherited disorders. DNA fingerprints helps to link suspects to biological evidence – blood or semen stains, hair, or items of clothing – found at the scene of a crime and help in solving crime. Another important use of DNA fingerprints in the court system is to establish paternity in custody and child support litigation. The U.S. armed services have just begun a program to collect DNA fingerprints from all personnel for use later, in case they are needed to identify casualties or persons missing in action or for suspect verification.

Due to the revolutionary development of biotechnology during last couple of decades agriculture has drastically advanced. Sensational achievements were made in both plant cultivation and animal husbandry. Plants have been improved in four different ways:

• Enhanced potential for more vigorous growth and increasing yields
• Increased resistance to natural predators and pests, including insects and disease-causing microorganisms.
• Production of hybrids exhibiting a combination of superior traits derived from two different strains or even different species
• Selection of genetic variants with desirable qualities such as increased protein value, increased content of limiting amino acids, which are essential in the human diet, or smaller plant size, reducing vulnerability to adverse weather condition.

Another important area of biotechnology is improvement of livestock. Improvement in disease control, efficiency of reproduction, yields of livestock products i.e. meat, milk, wool, eggs, composition of livestock products i.e. leaner meat, feed value of low quality feeds i.e. straw; are some of the applications of biotechnology.

One of the major scientific revolutions of the twentieth century was the breaking of the genetic code and the development of tools that enable scientists to probe the molecules of life with incredible precision. Now, in the twenty-first century, these developments in biology are being married with the use of ever-increasing computer power to help us face the challenges that the new century brings. Bioinformatics is the name given to the new discipline that has emerged at the interface of biology and computing. Huge amount of genetic data (DNA, RNA, amino acid and protein sequences) of various organisms, form bacteria to humans, being generated worldwide is stored in a computer database. Specialized software programs are used to find, visualize, and analyze the information, and most importantly, communicate it to other people. Various computer tools are used to predict protein structure which is a valuable information for development of vaccines, diagnostic tools as well as more effective drugs. Bioinformatics can help in easy and early detection of various diseases like cancer, diabetes and many more with the help of microarray chips (microarrays are miniature arrays of gene fragments attached to glass slides). Bioinformatics also helps scientists to construct phylogenetic tree based on molecular biology and ultimately contribute in the study of evolution. Computer simulations model such things as population dynamics, or calculate the cumulative genetic health of a breeding pool (in agriculture) or endangered population (in conservation). One very exciting potential of this field is that entire DNA sequences or genomes of endangered species can be preserved.

Biotechnology has a promising future. In future biotechnology will be accredited for some revolutionary technology. Recent advances in bioenergy, bioremediation, synthetic biology, DNA computers, virtual cell, genomics, proteomics, bioinformatics and bio-nanotechnology have made biotechnology even more powerful. Recent discovery of conduction of electricity by DNA and its behavior as a superconductor has opened a new realm in modern science. In future biotechnology will have profound impact in world economy. Biotechnology is a golden tool to solve some of the key global problems like global epidemic, fatal diseases, global warming, rising petroleum fuel crisis and above all poverty.

For all the positive effects of biotechnology there are some possible side effects. Nobody knows what ecological hazards could be caused by transgenic organisms. Some even speculate that some transgenic organisms could fall into wrong hands to develop bioweapons. The opposition of genetic engineering says that – the science is very young and needs a lot more research.

The path from a test tube to the field is not a straight highway. Both intellectual and financial resources should be realized before new discoveries pave their way to industrial applications. In conclusion, biotechnology has also proved to be extremely productive and innovative and 21st century should be the century of biotechnology.

Everything you wanted to know

Everything you wanted to know





Introduction

Sixty-five million years ago the dinosaurs died out along with more than 50% of other life forms on the planet. This mass extinction is so dramatic that for many years it was used to mark the boundary between the Cretaceous Period, when the last dinosaurs lived, and the Tertiary Period, when no dinosaurs remained. This is called the Cretaceous/Tertiary (or K/T) boundary, and the associated extinction is often termed the K/T extinction event.The name "Tertiary" is a holdover from the early days of geology, and many geologists now prefer the term "Paleogene" for the time period that immediately follows the Cretaceous. These scientists refer to the Cretaceous/Paleogene or K/P boundary, which represents the same moment in time as the K/T event. Since their discovery in the nineteenth century, the reason for the dinosaurs' demise has been a matter of speculation and debate. Early paleontologists, working prior to Darwin's theory of evolution by natural selection, suggested that dinosaurs represented the remains of animals that had perished in the Biblical Flood. This explained both the fact and speed of their disappearance. But as other extinctions came to light, and Darwin's theory gained acceptance, this explanation fell out of favor.

For many decades, the fossil record of dinosaurs was poorly known. During that time it was clear that dinosaurs had gone extinct, but it was not yet understood that this extinction was relatively sudden and simultaneous with those of many other species. Only at the end of the nineteenth century did paleontologists realize that nearly all dinosaurs had gone extinct within a brief period of time at the end of the Cretaceous Period.

For most of the next century, scientists focused on explanations for how the extinction might have occurred. Most theories focused on climate change, perhaps brought on by volcanism, lowering sea level, and shifting continents. But hundreds of other theories were developed, some reasonable but others rather far-fetched (including decimation by visiting aliens, widespread dinosaur "wars", and "paläoweltschmertz"­the idea that dinosaurs just got tired and went extinct). It was often popularly thought that the evolving mammals simply ate enough of the dinosaurs' eggs to drive them to extinction.

Regardless of the details, most of these theories shared the common thought that dinosaurs were a group of animals that had reached the end of their evolutionary life. Their extinction was seen as inevitable, the product of having evolved for too long. In most extinction scenarios, the dinosaurs were simply unable to cope with competition from mammals and the changing climate, and so they all went extinct.

As dinosaur science began to alter this hypothesis, producing a new view of dinosaurs as successful and viable organisms, many of these extinction theories became less tenable. New information from fossil localities suggested that many other organisms, most unrelated to dinosaurs, had also gone extinct at the same time. New theories were required to explain these new discoveries and newly understood facts. A favored theory was that tectonically induced climate change interfered with food chains, disrupting them enough to cause widespread extinction among many different organisms.

lvarez Hypothesis: Origin and Evidence
In the late 1970's geologist Walter Alvarez, and his father, Nobel-prize winning physicist Luis Alvarez, identified an unusual clay layer at the K/T boundary in Italy. This clay contained an unusually high concentration of the rare-earth element iridium ­ 30 times the level typically found in the Earth's crust. Why was the discovery of iridium so important? Although iridium is rare in the crust, it is abundant in many meteorites and asteroids as well as the Earth's core. With this evidence, Alvarez hypothesized that an asteroid must have struck the Earth right at the K/T boundary. Further investigation has revealed that this iridium-rich layer of clay occurs at more than 100 sites around the world, providing evidence that this was truly a worldwide event.

It was estimated that to produce the amount of iridium in the clay layer, the impact object would have been 10 km in diameter. Further evidence of an impact was discovered in the form of small grains of impact-shocked quartz and beads of impact glass (tektites) within the clay layer. Shocked quartz is formed by high-pressure shock waves, and is found at nuclear bomb sites and in meteor craters. Tektites are formed from the condensation of vaporized meteorite particles. Although shocked quartz has been found in K/T layers worldwide, tektites decrease in size with increasing distance from the impact site until they are altogether absent.

These pieces, along with high levels of iridium, provide evidence for an extraterrestrial impact at the end of the Cretaceous Period. Thus, the end of the dinosaurs’ reign may have been caused by an asteroid, not by sea level change or volcanism. Initially this theory was highly controversial, but today an extraterrestrial impact is considered to be a key factor in the K/T extinction event.

One of the main objections to the Alvarez theory was the absence of a 65-million-year-old crater anywhere on the Earth’s surface. Surely such an enormous asteroid impact would have left a sizable crater behind. In 1991, geologists discovered evidence for a huge crater at Chicxulub (pronounced CHIK-shoo-loob), on the Yucatan Peninsula in Mexico. Although the crater had long since been buried by hundreds of meters of sediment, surveys of magnetic and gravitational fields revealed its circular structure. In addition, recent sensitive topographic mapping has shown a low mound that represents part of the crater’s rim. At 180 km across, and dated to 65 mya, the crater is of the right size and age to have been caused by a 10 km asteroid hitting Earth at the end of the Cretaceous Period.

Effects of the Asteroid Impact
The devastation caused by such an event is difficult to imagine. The asteroid would have hit with the force of 100,000 billion tons of TNT. This would have generated an earthquake one thousand times greater than the largest ever recorded, with winds of over 400 kph. A massive fireball would have boiled nearby seas, destroying everything for thousands of kilometers. Forests throughout most of North America and some of South America would have been flattened by the shock wave. Evidence of a giant tsunami has been found around the Gulf of Mexico and Caribbean, as well as in Spain and Brazil. It may have had an effect as far away as New Zealand. 
Despite the enormity of the destruction from the initial impact, the dinosaurs and their contemporaries might have survived and eventually recovered, but the subsequent long-term effects of the blast were even more deadly. Ninety thousand cubic kilometers of debris would have been blasted into the atmosphere, some reaching into space only to re-enter at high speeds. This could have heated the atmosphere sufficiently to ignite global forest fires. While the heavier pieces of ejecta settled back down on Earth, fine dust particles would have remained in the atmosphere and significantly blocked sunlight, causing an effect called an “impact winter”. There is much debate about the duration and severity of the impact winter following the K/T impact, but the darkness and cold temperatures might have reduced photosynthesis and collapsed food chains globally.

The amount of carbon and sulfur contained in the rock at the impact site would have aggravated these devastating effects. As much as 100 billion tons of sulfur and 10 trillion tons of carbon would have been vaporized by the impact and blown into the atmosphere. The resulting sulfate aerosols would have stayed in the atmosphere for several years; the resulting carbon dioxide would have stayed airborne for several hundred years. Initially the sulfate aerosols would have contributed to global cooling by blocking out the sun, before precipitating as acid rain. After the dust and sulfates settled out and ended the cooling, global warming would have begun. The carbon dioxide levels, being two to three times normal, would have caused extreme greenhouse conditions, raising global temperatures by as much as 10°C. Although some life forms may have survived the years of darkness and freezing temperatures, many surely died out in the subsequent centuries of heat

Other Extinction Hypotheses
Although the impact hypothesis is the most widely accepted explanation of the K/T extinction, other theories still remain. Evidence of widespread volcanism, particularly at the Deccan traps in India, correlates with this moment in time as well. Prolonged volcanism could have led to atmospheric and climatic changes similar to those proposed for an asteroid impact. However, volcanism does not provide an alternate explanation for the high levels of iridium in the clay layer, because high concentrations of iridium occur deep in Earth's core rather than in the mantle, which is the source of the magma that was erupted.

One debate centers on whether the extinction was truly as sudden as it appears, or whether this is an artifact of the geological record. Some scientists believe that dinosaurs went extinct gradually, and were doing so for millions of years prior to the K/T boundary. Studies in the Western Interior of North America have suggested that the latest layers of Cretaceous sediments contain fewer dinosaur species than those below. These results have been challenged by other researchers, who claim that no such decrease is apparent in the Late Cretaceous record.

Deep-sea Evidence for the Impact Hypothesis
The general acceptance of the K/T asteroid impact theory has led many scientists to focus on the specific mechanisms that may have contributed to this dramatic extinction event. Although the impact was an important factor in the extinction of so many organisms, the event has also proven to be complex. In particular, the selectivity of the extinction has puzzled many paleontologists: why did dinosaurs go extinct but not crocodiles or turtles? Why did marine reptiles, belemnites, and ammonites disappear, but not fish or sharks? Why some mammals and not others?

Other scientists have focused on the extinction record preserved in deep-sea sediments in order to better understand the chain of events that followed the asteroid impact. Dr. Brian T. Huber, micropaleontologist in the National Museum of Natural History Dept. of Paleobiology, has studied evidence from a deep-sea drilling core taken 500-580 km of the northeastern coast of Florida during an Ocean Drilling Program cruise. Huber studied microscopic marine organisms called foraminifera taken from the core. The specimens were extracted from both Cretaceous and Tertiary age sediments. In one 40 cm core interval, he noticed a dramatic difference between the types of planktonic (floating) foraminifera that were alive prior to the boundary event and those that lived after. Prior to the extinction, large, ornate planktonic foraminifera were abundant, but afterward most specimens belonged to smaller, less ornate species. Overall more than 90% of the Cretaceous planktonic foraminifera had gone extinct. This is comparable to the extinction rate of calcareous nannofossils, another group of microscopic fossils that are abundant in the deep-sea sediment. In addition to the foraminifera, Huber also found specimens of shocked quartz and tektites, direct evidence of the impact itself.

The core also offered visual clues to the changes that occurred at the time of the extinction. The sediment undergoes a dramatic color change from white Cretaceous chalk in the lower portion of the core, to a dark gray, coarse-grained tektite layer in the middle, to a whitish gray Tertiary muddy chalk in the upper part. At the top of the tektite layer is a very thin, rust-colored, iron-rich layer known as the fireball layer. This rust layer, which has been found at a number of complete K/T impact horizons around the world, contains actual particles of the asteroid along with fine soot and ash that rained down on Earth's surface after the collision. This provides further evidence supporting the asteroid impact hypothesis.

Post-Extinction Recovery
It has been estimated that the planet took 1-2 million years to fully recover from the asteroid impact. In deep-sea sediments, several very small sized species of foraminifera with simple, unadorned shells appeared within several thousand years after the extinction event, but several million years elapsed before species diversity, shell ornamentation, and shell sizes increased to values comparable to those that occurred before the impact event. The small sized planktonic foraminifera are considered opportunistic species that had rapid rates of reproduction and higher tolerances to changing environmental conditions.

A similar pattern of extinction and recovery has been observed in the North American fossil land plant record. In southwestern North Dakota, where the fossil record of land plants is most complete and best studied, abrupt extinction of 70 to 90% of plant species was immediately followed by a dramatic increase in the abundance of ferns. Because the North American forests were decimated by the asteroid impact, ferns were able to rapidly disperse and dominate much of the newly cleared land surface for hundreds to thousands of years afterwards. Full recovery of North American forests, resulting from appearance of new species and repopulation by surviving species, took from several hundred thousand to over a million years.

Of the many long-term effects produced by the global devastation at the K/T boundary, the most obvious is the disappearance of all non-avian dinosaurs. Yet the close of the Age of Dinosaurs meant the start of the Age of Mammals. Although mammals had existed alongside the dinosaurs for hundreds of millions of years, they had remained small and comparatively rare. The extinction of the dinosaurs allowed mammals to come into dominance, as they evolved into new and larger forms throughout the Tertiary Period.

Within the first five million years of the Paleocene Epoch, large mammals had appeared for the first time. Some of them were the earliest members of modern groups, including primitive carnivorans and ungulates. The first primates (members of the mammalian order that includes humans) appeared about 10 million years after the K/T boundary event. Modern bird groups diversified as well, in the absence of pterosaurs (which had also gone extinct). Perhaps without the extinction of the dinosaurs, the evolution of mammals and the subsequent rise of humans would have never happened. And although recent history might well be called the Human Age, the time that the human race has dominated planet Earth is but a blink in geologic terms. It is certain that the world will change again. Indeed, we may be in the midst of another mass extinction event right now.

Your Facial Bone Structure Has a Big Influence on How People See You

Selfies, headshots, mug shots — photos of oneself convey more these days than snapshots ever did back in the Kodak era. Most digitally minded people continually post and update pictures of themselves at professional, social media and dating sites such as LinkedIn, Facebook, Match.com and Tinder. For better or worse, viewers then tend to make snap judgments about someone’s personality or character from a single shot. As such, it can be a stressful task to select the photo that conveys the best impression of ourselves. For those of us seeking to appear friendly and trustworthy to others, a new study underscores an old, chipper piece of advice: Put on a happy face.

A newly published series of experiments by cognitive neuroscientists at New York University is reinforcing the relevance of facial expressions to perceptions of characteristics such as trustworthiness and friendliness. More importantly, the research also revealed the unexpected finding that perceptions of abilities such as physical strength are not dependent on facial expressions but rather on facial bone structure.

The team’s first experiment featured photographs of 10 different people presenting five different facial expressions each. Study subjects rated how friendly, trustworthy or strong the person in each photo appeared. A separate group of subjects scored each face on an emotional scale from “very angry” to “very happy.” And three experts not involved in either of the previous two ratings to avoid confounding results calculated the facial width-to-height ratio for each face. An analysis revealed that participants generally ranked people with a happy expression as friendly and trustworthy but not those with angry expressions. Surprisingly, participants did not rank faces as indicative of physical strength based on facial expression but graded faces that were very broad as that of a strong individual.

In a second survey facial expression and facial structure were manipulated in computer-generated faces. Participants rated each face for the same traits as in the first survey, with the addition of a rating for warmth. Again, people thought a happy expression, but not an angry one, indicated friendliness, trustworthiness — and in this case, warmth. The researchers then showed two additional sets of participants the same faces, this time either with areas relevant to facial expressions obscured or the width cropped. In the first variation, for faces lacking emotional cues, people could no longer perceive personality traits but could still perceive strength based on width. Similarly, for those faces lacking structural cues, people could no longer perceive strength but could still perceive personality traits based on facial expressions.

In a third iteration of the survey participants had to pick four faces out of a lineup of eight faces varied for expression and width that they might select either as their financial advisor or as the winner of a power-lifting competition. As might be expected, participants picked faces with happier expressions as financial advisors and selected broader faces as belonging to power-lifting champs.

In a final survey the researchers generated more than 100 variations of one individual “base face” by varying facial features. Participants saw two faces at a time, and then picked one as either trustworthy or high in ability or as a good financial advisor or power-lifting winner. Using these results, a computer then created an average face for each of these four categories, which were shown to a separate set of participants who had to pick which face appeared either more trustworthy or stronger. Most of the participants found the computer-generated averages to be good representations of trustworthiness or strength — and generally saw the average “financial advisor” face as more trustworthy and the “power-lifter” face as stronger. The findings from all four surveys were published in the Personality and Social Psychology Bulletin on June 18.

Taken together the findings suggestion facial expressions strongly influence perception of traits such as trustworthiness, friendliness or warmth, but not ability (strength, in these experiments). Conversely, facial structure influences the perception of physical ability but not intentions (such as friendliness and trustworthiness, in this instance). In addition, decisions that involve guessing at the possible intentions of a person such as to whom you would entrust your money management are more strongly influenced by facial expression, whereas those based on physical ability such as whom you would bet on in a sporting event are more strongly influenced by facial structure.

Previous studies also have shown the effect of facial cues on how we perceive and interact with others but this new work reveals how perceptions of the same person can vary greatly depending on that person’s facial expression in any given moment. This variability “has implications for both the people presenting themselves and the perceivers in social interactions,” says Jonathan Freeman, a social neuroscientist at New York University and senior author of the study. So, we might consider the impact of our facial expressions in the photos we post online. At the same time, in an ideal world people who look at our photos would give us the benefit of the doubt and hesitate to make spontaneous judgments based only on a single image.

The findings above come with a big caveat: Only male faces were shown to subjects. The researchers chose this approach because previous studies involving the ratio of facial width to height have shown that greater facial width is often associated with higher testosterone levels as well as heightened aggression and strength in men. Studies of facial width and height in females have shown mixed results, so presenting study subjects with a mix of male and female faces would have yielded inconclusive results. Despite the relative lack of evidence on how facial structure influences perception of women’s faces, there have been humorous portrayals of popular speculations. Future research, however, is needed to definitively establish whether any such patterns exist.

Furthermore, the researchers refer to “ability” when discussing physical strength in the study. No specific measurements were made, for example, of perceptions of intellectual ability or ability to perform in certain job positions. These abilities are more abstract and thus might rely on a combination of different dynamic and static facial cues, Freeman explains, so it would be difficult to test these relationships definitively.

In our everyday lives this study and others make clear that although we might try to influence others’ perceptions of us with photos showing us donning sharp attire or displaying a self-assured attitude, the most important determinant of others' perception of and consequent behavior toward us is our faces.

So the next time you want to win someone’s trust, try a smile and a happy face. But for those folks hoping to get picked for a pick-up game of football, basketball and so on, don’t worry about your facial expression. The best you can do is hope you have a wider face and then let your physical prowess speak for itself.

Gene transformation

                                         Gene transformation

To achieve genetic transformation in plants, we need the construction of a vector (genetic vehicle) which transports the genes of interest, flanked by the necessary controlling sequences i.e. promoter and terminator, and deliver the genes into the host plant. The two kinds of gene transfer methods in plants are:

Vector-mediated or indirect gene transfer 

Among the various vectors used in plant transformation, the Ti plasmid of Agrobacterium tumefaciens has been widely used. This bacteria is known as “natural genetic engineer” of plants because these bacteria have natural ability to transfer T-DNA of their plasmids into plant genome upon infection of cells at the wound site and cause an unorganized growth of a cell mass known as crown gall. Ti plasmids are used as gene vectors for delivering useful foreign genes into target plant cells and tissues. The foreign gene is cloned in the T-DNA region of Ti-plasmid in place of unwanted sequences.
To transform plants, leaf discs (in case of dicots) or embryogenic callus (in case of monocots) are collected and infected with Agrobacterium carrying recombinant disarmed Ti-plasmid vector. The infected tissue is then cultured (co-cultivation) on shoot regeneration medium for 2-3 days during which time the transfer of T-DNA along with foreign genes takes place. After this, the transformed tissues (leaf discs/calli) are transferred onto selection cum plant regeneration medium supplemented with usually lethal concentration of an antibiotic to selectively eliminate non-transformed tissues. After 3-5 weeks, the regenerated shoots (from leaf discs) are transferred to root-inducing medium, and after another 3-4 weeks, complete plants are transferred to soil following the hardening (acclimatization) of regenerated plants. The molecular techniques like PCR and southern hybridization are used to detect the presence of foreign genes in the transgenic plants.

Vectorless or direct gene transfer

In the direct gene transfer methods, the foreign gene of interest is delivered into the host plant cell without the help of a vector. The methods used for direct gene transfer in plants are:

Chemical mediated gene transfer e.g. chemicals like polyethylene glycol (PEG) and dextran sulphate induce DNA uptake into plant protoplasts.Calcium phosphate is also used to transfer DNA into cultured cells.

Microinjection where the DNA is directly injected into plant protoplasts or cells (specifically into the nucleus or cytoplasm) using fine tipped (0.5 - 1.0 micrometerdiameter) glass needle or micropipette. This method of gene transfer is used to introduce DNA into large cells such as oocytes, eggs, and the cells of early embryo.

Electroporation involves a pulse of high voltage applied to protoplasts/cells/ tissues to make transient (temporary) pores in the plasma membrane which facilitates the uptake of foreign DNA.
The cells are placed in a solution containing DNA and subjected to electrical shocks to cause holes in the membranes. The foreign DNA fragments enter through the holes into the cytoplasm and then to nucleus.
Particle gun/Particle bombardment - In this method, the foreign DNA containing the genes to be transferred is coated onto the surface of minute gold or tungsten particles (1-3 micrometers) and bombarded onto the target tissue or cells using a particle gun (also called as gene gun/shot gun/microprojectile gun).The microprojectile bombardment method was initially named as biolistics by its inventor Sanford (1988). Two types of plant tissue are commonly used for particle bombardment- Primary explants and the proliferating embryonic tissues.
Transformation - This method is used for introducing foreign DNA into bacterial cells e.g. E. Coli. The transformation frequency (the fraction of cell population that can be transferred) is very good in this method. E.g. the uptake of plasmid DNA by E. coli is carried out in ice cold CaCl2 (0-50C) followed by heat shock treatment at 37-450C for about 90 sec. The transformation efficiency refers to the number of transformants per microgram of added DNA. The CaCl2 breaks the cell wall at certain regions and binds the DNA to the cell surface.
Conjuction - It is a natural microbial recombination process and is used as a method for gene transfer. In conjuction, two live bacteria come together and the single stranded DNA is transferred via cytoplasmic bridges from the donor bacteria to the recipient bacteria.

Liposome mediated gene transfer or Lipofection - Liposomes are circular lipid molecules with an aqueous interior that can carry nucleic acids. Liposomes encapsulate the DNA fragments and then adher to the cell membranes and fuse with them to transfer DNA fragments. Thus, the DNA enters the cell and then to the nucleus. Lipofection is a very efficient technique used to transfer genes in bacterial, animal and plant cells.

Selection of transformed cells from untransformed cells

The selection of transformed plant cells from untransformed cells is an important step in the plant genetic engineering. For this, a marker gene (e.g. for antibiotic resistance) is introduced into the plant along with the transgene followed by the selection of an appropriate selection medium (containing the antibiotic). The segregation and stability of the transgene integration and expression in the subsequent generations can be studied by genetic and molecular analyses (Northern, Southern, Western blot, PCR).

During the last decades, a tremendous progress has been made in the development of transgenic plants using the various techniques of genetic engineering. The plants, in which a functional foreign gene has been incorporated by any biotechnological methods that generally are not present in the plant, are called transgenic plants. As per estimates recorded in 2002, transgenic crops are cultivated world-wide on about 148 million acres (587 million hectares) land by about 5.5 million farmers. Transgenic plants have many beneficial traits like insect resistance, herbicide tolerance, delayed fruit ripening, improved oil quality, weed control etc.
Some of the commercially grown transgenic plants in developed countries are: “Roundup Ready” soybean, ‘Freedom II squash’, ‘High- lauric’ rapeseed (canola), ‘Flavr Savr’ and ‘Endless Summer’ tomatoes. During 1995, full registration was granted to genetically engineered Bt gene containing insect resistant ‘New Leaf’ (potato), ‘Maximizer’ (corn), ‘BollGard’ (cotton) in USA. Some of the traits introduced in these transgenic plants are as follows:

Stress tolerance

Biotechnology strategies are being developed to overcome problems caused due to biotic stresses (viral, bacterial infections, pests and weeds) and abiotic stresses (physical actors such as temperature, humidity, salinity etc).

Abiotic stress tolerance

The plants show their abiotic stress response reactions by the production of stress related osmolytes like sugars (e.g. trehalose and fructans), sugar alcohols (e.g. mannitol), amino acids (e.g. proline, glycine, betaine) and certain proteins (e.g. antifreeze proteins). Transgenic plants have been produced which over express the genes for one or more of the above mentioned compounds. Such plants show increased tolerance to environmental stresses. Resistance to abiotic stresses includes stress induced by herbicides, temperature (heat, chilling, freezing), drought, salinity, ozone and intense light. These environmental stresses result in the destruction, deterioration of crop plants which leads to low crop productivity. Several strategies have been used and developed to build ressitance in the plants against these stresses.

Herbicide tolerance

Weeds are unwanted plants which decrease the crop yields and by competing with crop plants for light, water and nutrients. Several biotechnological strategies for weed control are being used e.g. the over-production of herbicide target enzyme (usually in the chloroplast) in the plant which makes the plant insensitive to the herbicide. This is done by the introduction of a modified gene that encodes for a resistant form of the enzyme targeted by the herbicide in weeds and crop plants. Roundup Ready crop plants tolerant to herbicide-Roundup, is already being used commercially.
The biological manipulations using genetic engineering to develop herbicide resistant plants are: (a) over-expression of the target protein by integrating multiple copies of the gene or by using a strong promoter., (b) enhancing the plant detoxification system which helps in reducing the effect of herbicide., (c) detoxifying the herbicide by using a foreign gene., and (d) modification of the target protein by mutation.

Some of the examples are:

Glyphosate resistance - Glyphosate is a glycine derivative and is a herbicide which is found to be effective against the 76 of the world’s worst 78 weeds. It kills the plant by being the competitive inhibitor of the enzyme 5-enoyl-pyruvylshikimate 3- phosphate synthase (EPSPS) in the shikimic acid pathway. Due to it’s structural similarity with the substrate phosphoenol pyruvate, glyphosate binds more tightly with EPSPS and thus blocks the shikimic acid pathway.
Certain strategies were used to provide glyphosate resistance to plants.

(a) It was found that EPSPS gene was overexpressed in Petunia due to gene amplification. EPSPS gene was isolated from Petunia
and introduced in to the other plants. These plants could tolerate glyphosate at a dose of 2- 4 times higher than that required to kill wild type plants.

(a) By using mutant EPSPS genes- A single base substitution from C to T resulted in the change of an amino acid from proline to serine in EPSPS. The modified enzyme cannot bind to glyphosate and thus provides resistance.

(b) The detoxification of glyphosate by introducing the gene (isolated from soil organism- Ochrobactrum anthropi) encoding for glyphosate oxidase into crop plants. The enzyme glyphosate oxidase converts glyphosate to glyoxylate and aminomethylphosponic acid. The transgenic plants exhibited very good glyphosate ressitance in the field.

Another example is of Phosphinothricin resistance

Phosphinothricin is a broad spectrum herbicide and is effective against broad-leafed weeds. It acts as a competitive inhibitor
of the enzyme glutamine synthase which results in the inhibition of the enzyme glutamine synthase and accumulation of ammonia and finally the death of the plant. The disturbace in the glutamine synthesis also inhibits the photosynthetic activity.
The enzyme phosphinothricin acetyl transferase ( which was first observed in Streptomyces sp in natural detoxifying mechanism against phosphinothricin) acetylates phosphinothricin, and thus inactivates the herbicide. The gene encoding for phosphinothricin acetyl transferase (bar gene) was introduced in transgenic maize and oil seed rape to provide resistance against phosphinothricin.

Other abiotic stresses

The abiotic stresses due to temperature, drought, and salinity are collectively also known as water deficit stresses. The plants produce osmolytes or osmoprotectants to overcome the osmotic stress. The attempts are on to use genetic engineering strategies to increase the production of osmoprotectants in the plants. The biosynthetic pathways for the production of many osmoprotectants have been established and genes coding the key enzymes have been isolated. E.g. Glycine betaine is a cellular osmolyte which is produced by the participation of a number of key enzymes like choline dehydrogenase, choline monooxygenase etc. The choline oxidase gene from Arthrobacter sp. was used to produce transgenic rice with high levels of glycine betaine giving tolerance against water deficit stress.
Scientists also developed cold-tolerant genes (around 20) in Arabidopsis when this plant was gradually exposed to slowly declining temperature. By introducing the coordinating gene (it encodes a protein which acts as transcription factor for regulating the expression of cold tolerant genes), expression of cold tolerant genes was triggered giving protection to the plants against the cold temperatures. 

Insect resistance

A variety of insects, mites and nematodes significantly reduce the yield and quality of the crop plants. The conventional method is to use synthetic pesticides, which also have severe effects on human health and environment. The transgenic technology uses an innovative and eco-friendly method to improve pest control management.About 40 genes obtained from microorganisms of higher plants and animals have been used to provide insect resistance in crop plants

The first genes available for genetic engineering of crop plants for pest resistance were Cry genes (popularly known as Bt genes) from a bacterium Bacillus thuringiensis. These are specific to particular group of insect pests, and are not harmful to other useful insects like butter flies and silk worms. Transgenic crops with Bt genes (e.g. cotton, rice, maize, potato, tomato, brinjal, cauliflower, cabbage, etc.) have been developed. This has proved to be an effective way of controlling the insect pests and has reduced the pesticide use. The most notable example is Bt cotton (which contains CrylAc gene) that is resistant to a notorious insect pest Bollworm (Helicoperpa armigera).. There are certain other insect resistant genes from other microorganisms which have been used for this purpose. Isopentenyl transferase gene from Agrobacterium tumefaciens has been introduced into tobacco and tomato. The transenic plants with this transgene were found to reduce the leaf consumption by tobacco hornworm and decrease the survival of peach potato aphid.

Certain genes from higher plants were also found to result in the synthesis of products possessing insecticidal activity. One of the examples is the Cowpea trypsin inhibitor gene (CpTi) which was introduced into tobacco, potato, and oilseed rape for develping transgenic plants. Earlier it was observed that the wild species of cowpea plants growing in Africa were resistant to attack by a wide range of insects. It was observed that the insecticidal protein was a trypsin inhibitor that was capable of destroying insects belonging to the orders Lepidoptera, Orthaptera etc. Cowpea trypsin inhibitor (CpTi) has no effect on mammalian trypsin, hence it is non-toxic to mammals. 

Virus resistance

There are several strategies for engineering plants for viral resistance, and these utilizes the genes from virus itself (e.g. the viral coat protein gene). The virus-derived resistance has given promising results in a number of crop plants such as tobacco, tomato, potato, alfalfa, and papaya. The induction of virus resistance is done by employing virus-encoded genes-virus coat proteins, movement proteins, transmission proteins, satellite RNa, antisense RNAs, and ribozymes. The virus coat protein-mediated approach is the most successful one to provide virus resistance to plants. It was in 1986, transgenic tobacco plants expressing tobacco mosaic virus (TMV) coat protein gene were first developed. These plants exhibited high levels of resistance to TMV.
The transgenic plant providing coat protein-mediated resistance to virus are rice, potato, peanut, sugar beet, alfalfa etc. The viruses that have been used include alfalfa mosaic virus (AIMV), cucumber mosaic virus (CMV), potato virus X (PVX) , potato virus Y (PVY) etc.

Resistance against Fungal and bacterial infections

As a defense strategy against the invading pathogens (fungi and bacteria) the plants accumulate low molecular weight proteins which are collectively known as pathogenesis-related (PR) proteins.
Several transgenic crop plants with increased resistance to fungal pathogens are being raised with genes coding for the different compounds. One of the examples is the Glucanase enzyme that degrades the cell wall of many fungi. The most widely used glucanase is beta-1,4-glucanase. The gene encoding for beta-1,4 glucanase has been isolated from barley, introduced, and expressed in transgenic tobacco plants. This gene provided good protection against soil-borne fungal pathogen Rhizoctonia solani.
Lysozyme degrades chitin and peptidoglycan of cell wall, and in this way fungal infection can be reduced. Transgenic potato plants with lysozyme gene providing resistance to Eswinia carotovora have been developed. 

Delayed fruit ripening

The gas hormone, ethylene regulates the ripening of fruits, therefore, ripening can be slowed down by blocking or reducing ethylene production. This can be achieved by introducing ethylene forming gene(s) in a way that will suppress its own expression in the crop plant. Such fruits ripen very slowly (however, they can be ripen by ethylene application) and this helps in exporting the fruits to longer distances without spoilage due to longer-shelf life.
The most common example is the 'Flavr Savr' transgenic tomatoes, which were commercialized in U.S.A in 1994. The main strategy used was the antisense RNA approach. In the normal tomato plant, the PG gene (for the enzyme polygalacturonase) encodes a normal mRNA that produces the enzyme polygalacturonase which is involved in the fruit ripening. The complimentary DNA of PG encodes for antisense mRNA, which is complimentary to normal (sense) mRNA. The hybridization between the sense and antisnse mRNAs renders the sense mRNA ineffective. Consequently, polygalacturonase is not produced causing delay in the fruit ripening. Similarly strategies have been developed to block the ethylene biosynthesis thereby reducing the fruit ripening. E.g. transgenic plants with antisense gene of ACC oxidase (an enzyme involved in the biosynthetic process of ethylene) have been developed. In these plants, production of ethylene was reduced by about 97% with a significant delay in the fruit ripening.
The bacterial gene encoding ACC deaminase (an enzyme that acts on ACC and removes amino group) has been transferred and expressed in tomato plants which showed 90% inhibition in the ethylene biosynthesis. 

Male Sterility

The plants may inherit male sterility either from the nucleus or cytoplasm. It is possible to introduce male sterility through genetic manipulations while the female plants maintain fertility. In tobacco plants, these are created by introducing a gene coding for an enzyme (barnase, which is a RNA hydrolyzing enzyme) that inhibits pollen formation. This gene is expressed specifically in the tapetal cells of anther using tapetal specific promoter TA29 to restrict its activity only to the cells involved in pollen production. The restoration of male fertility is done by introducing another gene barstar that suppresses the activity of barnase at the onset of the breeding season. By using this approach, transgenic plants of tobacco, cauliflower, cotton, tomato, corn, lettuce etc. with male sterility have been developed.

Plants can be used as cheap chemical factories that require only water, minerals, sun light and carbon dioxide to produce thousands of sophisticated chemical molecules with different structures. By transferring the right genes, plants can serve as bioreactors to modified or new compounds such as amino acids, proteins, vitamins, plastics, pharmaceuticals (peptides and proteins), drugs, enzymes for food industry and so on. The transgenic plants as bioreactors have some advantages such as the cost of production is low, there is an unlimited supply, safe and environmental friendly and there is no scare of spread of animal borne diseases.
Tobacco is the most preferred plant as a transgenic bioreactor because it can be easily transformed and engineered. Tobacco is an excellent biomass producer with about 40 tons of fresh leaf production as against e.g. rice with 4 tons. The seed production is very high (approx. one million seeds per plant) and it can be harvested several times in a year.
Some of the uses of transgenic plants are:

Improvement of Nutrient quality

Transgenic crops with improved nutritional quality have already been produced by introducing genes involved in the metabolism of vitamins, minerals and amino acids.
A transgenic Arabidopsis thaliana that can produce ten-fold higher vitamin E (alpha-tocopherol) than the native plant has been developed. The biochemical machinery to produce a compound close in structure to alpha-tocopherol is present in A. thaliana. A gene that can finally produce alpha-tocopherol is also present, but is not expressed. This dormant gene was activated by inserting a regulatory gene from a bacterium which resulted in an efficient production of vitamin E.
Glycinin is a lysine-rich protein of soybean and the gene encoding glycinin has been introduced into rice and successfully expressed. The transgenic rice plants produced glycinin with high contents of lysine.
Using genetic engineering Prof Potrykus and Dr. Peter Beyer have developed rice which is enriched in pro-vitamin A by introducing three genes involved in the biosynthetic pathway for carotenoid, the precursor for vitamin A. The aim was to help millions of people who suffer from night blindness due to Vitamin A deficiency, especially whose staple diet is rice. The presence of beta-carotene in the rice gives a characteristic yellow/orange colour, hence this pro-vitamin A enriched rice is named as Golden Rice.
The genetic engineering is also being used to improve the taste of food e.g. a protein ‘monellin’ isolated from an African plant (Dioscorephyllum cumminsii) is about 100,000 sweeter than sucrose on molar basis. Monellin gene has been introduced into tomato and lettuce plants to improve their taste.

Improvement of seed protein quality

The nutritional quality of cereals and legumes has been improved by using biotechnological methods. Two genetic engineering approaches have been used to improve the seed protein quality. In the first case, a transgene (e.g. gene for protein containing sulphur rich amino acids) was introduced into pea plant (which is deficient in methionine and cysteine, but rich in lysine) under the control of seed-specific promoter. In the second approach, the endogenous genes are modified so as to increase the essential amino acids like lysine in the seed proteins of cereals.
These transgenic routes have helped to improve the essential amino acids contents in the seed storage proteins of a number of crop plants. E.g. overproduction of lysine by de-regulation. The four essential amino acids namely lysine, methionine, threonine, and isoleucine are produced from a non-essential amino acid aspartic acid. The formation of lysine is regulated by feed back inhibition of the enzymes aspartokinase (AK) and dihydrodipicolinate synthase (DHDPS). The lysine feedback- insensitive genes encoding the enzymes AK and DHDPS have been respectively isolated from E. Coli and Cornynebacterium. After doing appropriate genetic manipulations, these genes were introduced into soybean and canola plants. The transgenic plants so produced had high quantities of lysine.

Diagnostic and therapeutic proteins

Experiments are going on to use transgenic plants in diagnostics for detecting human diseases and therapeutics for curing human and animal diseases. Several metabolites and compounds are already being produced in transgenic plants e.g. the monoclonal antibodies, blood plasma proteins, peptide hormones, cytokinins etc. The use of plants for commercial production of antibodies, referred to as plantbodies, is a novel approach in biotechnology. The first successful production of a functional antibody, namely a mouse immunoglobulin IgGI in plants, was reported in 1989. This was achieved by developing two transgenic tobacco plants-one synthesizing heavy chain gamma- chain and other light kappa- chain, and crossing them to generate progeny that can produce an assembled functional antibody. In 1992, C.J. Amtzen and co-workers expressed hepatitis B surface antigen in tobacco to produce immunologically active ingredients via genetic engineering of plants.
Several other therapeutic proteins have also been produced like haemoglobin and erythropoietin in tobacco plants, lactoferrin in potato, trypsin inhivitor in maize etc. The first proteins/enzymes that were produced in transgenic plants (maize) are avidin and beta-glucuronidase and are used in diagnostic kits. 

Edible vaccines

Crop plants offer cost-effective bioreactors to express antigens which can be used as edible vaccines. The approach is to isolate genes encoding antigenic proteins from the pathogens and then expressing them in plants. Such transgenic plants or their tissues producing antigens can be eaten for vaccination/immunization (edible vaccines). The expression of such antigenic proteins in crops like banana and tomato are useful for immunization of humans since banana and tomato fruits can be eaten raw.
Transgenic plants (tomato, potato) have been developed for expressing antigens derived from animal viruses e.g. rabies virus, herpes virus. In 1990, the first report of the production of edible vaccine (a surface protein from Streptococcus) in tobacco at 0.02% of total leaf protein level was published in the form of a patent application under the International Patent Cooperation Treaty (Mason and Arntzen,1995).The first clinical trials in humans, using a plant derived vaccine were conducted in 1997 and were met with limited success. This involved the ingestion of transgenic potatoes with a toxin of E. coli causing diarrhea.

The process of making of edible vaccines involves the incorporation of a plasmid carrying the antigen gene and an antibiotic resistance gene, into the bacterial cells e.g. Agrobacterium tumefaciens. The small pieces of potato leaves are exposed to an antibiotic which can kill the cells that lack the new genes. The surviving cells with altered genes multiply and form a callus. This callus is allowed to grow and subsequently transferred to soil to form a complete plant. In about a few weeks, the plants bear potatoes with antigen vaccines.

The bacteria E.coli, V. cholerae cause acute watery diarrhea by colonizing the small intestine and by producing toxins. Chloera toxin (CT) is very similar to E.Coli toxin. The CT has two subunits, A and B. Attempt was made to produce edible vaccine by expressing heat labile enterotoxin (CT-B) in tobacco and potato.

Another strategy adopted to produce a plant-based vaccine, is to infect the plants with recombinant virus carrying the desired antigen that is fused to viral coat protein. The infected plants are reported to produce the desired fusion protein in large amounts in a short duration. The technique involves either placing the gene downstream a subgenomic promoter, or fusing the gene with capsid protein that coats the virus.

Advantages of edible vaccines

The edible vaccines produced in transgenic plants will sole the storage problems, will ensure easy delivery system by feeding and will have low cost as compared to the recombinant vaccines produced by bacterial fermentation. Vaccinating people against dreadful diseases like cholera and hepatitis B, by feeding them banana, tomato, and vaccinating animals against important diseases will be an interesting development.

Biodegradable plastics

Polythenes and plastics are one of the major environmental hazards. Efforts are on to explore the possibility of using transgenic plants for biodegradable plastics. Transgenic plants can be used as factories to produce biodegradable plastics like polyhydroxy butyrate or PHB. Genetically engineeredArabidopisis plants can produce PHB globules exclusively in their chloroplasts without effecting plant growth and development. The large-scale production of PHB can easily be achieved in plants likePopulus, where PHB can be extracted from leaves.

Molecular Breeding
The term molecular breeding is frequently used to represent the breeding methods that are coupled with genetic engineering techniques. Up till now, conventional breeding methods have been used to meet the food demands of the growing world population and the challenges of poverty and improved crop production and yields. However in the years to come, the development in the agriculture yields and techniques is going to be due to the use of molecular breeding programme.
Linkage analysis which deals with the studies to correlate the link between the molecular marker and a desired trait is an important aspect of molecular breeding programme. In the past, linkage analysis was carried out by use of isoenzymes and the associated polymorphisms. Now a days, molecular markers are being used.

Molecular breeding involves breeding using molecular (nucleic acid) markers. A molecular marker is a DNA sequence in the genome which can be located and identified therefore molecular markers can be used to identify particular locations in the genome.

Due to mutations, insertions, deletions, etc. the base composition at a particular location may be different in different plants. These differences, termed polymorphisms, allow DNA markers to be mapped in a genetic linkage group.
Generally, there are three types of markers used in screening/selection:

a) Morphological marker based on visible character (phenotypic expression) e.g. flower color, seed color, height, leaf shapes, etc. Morphological markers could be dominant or recessive. There are certain constraints in using these markers as the morphological markers are easily influenced by environmental factors and thus may not represent the desired genetic variation. Some of the visible markers have not much role to play in the plant breeding programme.

b) Biochemical marker: The proteins produced by gene expression are also used as markers in plant breeding programmes. The most commonly used are isozymes, the different molecular forms of the same enzyme. Each individual variety has its own isozyme variability (profiles) which can be detected by electrophoresis on starch gel.
c) Molecular marker based on DNA polymorphism detected by DNA probes or amplified products of PCR, e.g.Restriction fragment length polymorphism (RFLP), Randomly Amplified polymorphic DNA (RAPD), variable Number Tandom Repeats (VNTR), Microsatellites, etc. Plant breeders always prefer to detect the gene as molecular marker, although it is not always possible. Molecular markers provide a true representation of the genetic make up at the DNA level. They are consistent and free from environmental factors, and can be detected much before the development of plants occur. The advantage with a molecular marker is that a plant breeder can select a suitable marker for the desired trait which can be detected well in advance. A large number of markers can be generated as per the needs. The molecular markers to be used in plant breeding programme should have the following characteristics: (a) the marker should be closely linked with the desired trait, (b) the marker screening methods should be effective, efficient, reproducible and easy to carry out, (C) the entire analysis should be cost effective.

Molecular makers are of two types: (a) based on nucleic acid (DNA) hybridization- This involves the cloning of the DNA piece followed by the hybridization with the genomic DNA, which is later detected.
The Restriction fragment length polymorphism (RFLP) was the very first technology employed for the detection of polymorphism, based on the DNA sequence differences. RFLP is mainly based on the altered restriction enzyme sites, as a result of mutations and recombinations of genomic DNA. The procedure involves the isolation of genomic DNA and it’s digestion by restriction enzymes. The fragments are separated by electrophoresis and finally hybridized by incubating with cloned and labeled probes.

(b) Molecular markers based on PCR amplification.
Polymerase chain reaction (PCR) is a novel technique for the amplification of selected regions of DNA. The most important advantage is that even a minute quantity of DNA can be amplified and the PCR- based molecular markers require only a small quantity of DNA to start with. Random amplified polymorphic DNA (RAPD) markers use PCR amplification where the DNA is isolated from the genome and is denatured. The template molecules are annealed with primers and amplified by PCR. The amplified products are separated on electrophoresis and identified. Based on the nucleotide alterations in the genome, the polymorphisms of amplified DNA sequences differ which can be identified as bends on gel electrophoresis.
Amplified fragment length polymorphism (AFLP) is a novel technique involving a combination of RFLP and RAPD. AFLP is based on the principle of generation of DNA fragments using restriction enzymes and oligonucleotide adaptors (or linkers), and their amplification by PCR.

Microsatellites
Microsatellites are the tandemly repeated multiple copies of mono-, di-, tri-, and tetra nucleotide motifs. In some instances, there are unique flanking sequences present in the repeat sequences. Primers are designed for such flanking sequences to detect the sequence tagged microsatellites (STMS) which is done by PCR.

Commercial use of transgenic plants
The main goal of producing transgenic plants is to increase the productivity. In 1995-96, transgenic potato and cotton plants were used commercially for the first time in USA. By the year 1998-99, five other major transgenic crops cotton, maize, canola, soybean, and potato were introduced to the farmers. These accounted for about 75% of the total area planted by crops in USA. There are still a lot of concerns regarding the harmful environmental and hazardous health effects of transgenic plants. The major areas of public concern are- the development of resistance genes in insects, generation of a super weeds by mutation etc. Certain other legal and regulatory hurdles pertaining to commercial use of transgenic plants, needs to be addressed.

Bioethics in Plant genetic Engineering
There are issues and concerns regarding the use of transgenic crops and their effects on the health and the environment in general. The major concerns about GM crops and GM foods are:

a) Effect of GM crops on biodiversity and environment- As the GM crops are created artificially, there is no natural process of evolution in their development. Hence, there is a question of this affecting the biodiversity and overall effect on the environment.

b) The risk of transfer of transgene from GM crops to pathogenic microbes- Antibiotic marker genes are used to identify and select the modified cells. If GM food containing antibiotic resistance marker gene is consumed by animals and humans, there is a risk that the transgene will transfer from GM food to microflora of human and animals. This may lead to the gut microbes to become resistant to antibiotics.

c) The transfer of genes from animals into Gm crops for molecular farming may change the fundamental vegetable nature of plants.

d) The GM crops may bring about changes in evolutionary patterns. The plants adapt to the changing environment in the natural way by changing their genes and developing better races with superior traits which ultimately leads to the development of evolved races and varieties. What will be the evolutionary pattern of the GM crops? There are concerns about the effect of transgene flow from GM crops to other non-GM plants and the alteration of these non-GM crops.

e) There is a risk of transferring allergens (usually glycoproteins) from GM food to human and animals.

f) There is a risk of “gene pollution” i.e. transfer of transgene of GM crop through pollen grains to related plant species and development of super weeds.

g) There are also some religious issues related to the consumption of transgenic plants with animal genes introduced into them, especially, for some strict vegetarian people and some ethnic groups with certain food preferences and restrictions.

h) There is a need to study thoroughly as to how the genetically engineered plants will affect the ecological balance, once they are released in the environment.