ABT 211: INTRODUCTION TO PLANT BIOTECHNOLOGY

ABT 211: INTRODUCTION TO PLANT BIOTECHNOLOGY

1. INTRODUCTION

The word "biotechnology" was first used in 1917 to describe processes using living organisms to make a product or run a process, such as industrial fermentations. In fact, biotechnology began when humans started to plant their own crops, domesticate animals, ferment juice into wine, make cheese, and leaven bread.

Present definition of biotechnology

“Any technological application that uses biological systems, living organisms, or derivatives theory, to make or modify products or processes for specific use’’ (According to the Convention on Biological Diversity)

Plant biotechnology

“Plant biotechnology describes a precise process in which scientific techniques are used to develop useful and beneficial plants’’ (According to the Council for Biotechnology Information)

Traditional Biotechnology

Traditional biotechnology refers to a number of ancient ways of using living organisms to make new products or modify existing ones. In its broadest definition, traditional biotechnology can be traced back to human's transition from hunter-gatherer to farmer. As farmers, humans collected wild plants and cultivated them and the best yielding strains were selected for growing the following seasons.

As humans discovered more plant varieties and traits or characteristics, they gradually became adept at breeding specific plant varieties over several years and sometimes generations, to obtain desired traits such as disease resistance, better taste and higher yield. With the domestication of animals, ancient farmers applied the same breeding techniques to obtain desired traits among animals over generations.

Centuries ago, people accidentally discovered how to make use of natural processes that occur all the time within living cells. Although they had no scientific explanation for the processes, they applied the results they saw to their domestic lives. They discovered, for example, that food matures in a way that changes its taste and content, and makes it less perishable. Hence, through a process later called fermentation, flour dough becomes leavened in the making of bread, grape juice becomes wine, and milk stored in bags made from camels' stomachs turns into cheese.

Through trial and error and later through advances in technology, people learned to control these processes and make large quantities of biotechnology products. Advances in science enabled the transfer of these mostly domestic techniques into industrial applications and the discovery of new techniques. Examples of traditional biotechnology techniques include selective breeding, hybridization and fermentation.

Modern Biotechnology

Modern biotechnology refers to a number of techniques that involve the intentional manipulation of genes, cells and living tissue in a predictable and controlled manner to generate changes in the genetic make-up of an organism or produce new tissue. Examples of these techniques include: recombinant DNA techniques (rDNA or genetic engineering), tissue culture and mutagenesis.
Modern biotechnology began with the 1953 discovery of the structure of deoxyribonucleic acid (DNA) and the way genetic information is passed from generation to generation. This discovery was made possible by the earlier discovery of genes (discrete, independent units that transmit traits from parents to offspring) by Gregor Mendel. These discoveries laid the groundwork for the transition from traditional to modern biotechnology. They made it possible to produce desired changes in an organism through the direct manipulation of its genes in a controlled and less time-consuming fashion in comparison to traditional biotechnology techniques. These discoveries, coupled with advances in technology and science (such as biochemistry and physiology), opened up the possibilities for new applications of biotechnology which were unknown with traditional forms.

How does modern biotechnology work?

All organisms are made up of cells that are programmed by the same basic genetic material, called DNA (deoxyribonucleic acid).  Each unit of DNA is made up of a combination of the following nucleotides -- adenine (A), guanine (G), thymine (T), and cytosine (C) -- as well as a sugar and a phosphate.  These nucleotides pair up into strands that twist together into a spiral structure call a "double helix."  This double helix is DNA.  Segments of the DNA tell individual cells how to produce specific proteins.  These segments are genes.  It is the presence or absence of the specific protein that gives an organism a trait or characteristic. More than 10,000 different genes are found in most plant and animal species. This total set of genes for an organism is organized into chromosomes within the cell nucleus. The process by which a multicellular organism develops from a single cell through an embryo stage into an adult is ultimately controlled by the genetic information of the cell, as well as interaction of genes and gene products with environmental factors.

When cells reproduce, the DNA strands of the double helix separate. Because nucleotide A always pairs with T and G always pairs with C, each DNA strand serves as a precise blueprint for a specific protein. Except for mutations or mistakes in the replication process, a single cell is equipped with the information to replicate into millions of identical cells. Because all organisms are made up of the same type of genetic material (nucleotides A, T, G, and C), biotechnologists use enzymes to cut and remove DNA segments from one organism and recombine it with DNA in another organism. This is called recombinant DNA (rDNA) technology, and it is one of the basic tools of modern biotechnology. rDNA technology is the laboratory manipulation of DNA in which DNA, or fragments of DNA from different sources, are cut and recombined using enzymes. This recombinant DNA is then inserted into a living organism. rDNA technology is usually used synonymously with genetic engineering. rDNA technology allows researchers to move genetic information between unrelated organisms to produce desired products or characteristics or to eliminate undesirable characteristics.

Genetic engineering is the technique of removing, modifying or adding genes to a DNA molecule in order to change the information it contains. By changing this information, genetic engineering changes the type or amount of proteins an organism is capable of producing. Genetic engineering is used in the production of drugs, human gene therapy, and the development of improved plants. For example, an “insect protection” gene (Bt) has been inserted into several crops - corn, cotton, and potatoes - to give farmers new tools for integrated pest management. Bt corn is resistant to European corn borer. This inherent resistance thus reduces a farmer’s pesticide use for controlling European corn borer, and in turn requires fewer chemicals and potentially provides higher yielding Agricultural Biotechnology.

                           

2. IMPORTANCE OF BIOTECNOLOGY

3. CROP IMPROVEMENT

Conventional approaches in plant breeding involve reshuffling of genes among individuals falling within the sexually crossable groups. The in­volvement of genes under manipulation is usually inferred from their phenotypic effect on plants. But the new tools of molecular biology have broadened the scope of gene manipulations at the level of specific DNA segments across wide range of organisms to produce novel genomes with enhanced levels of resistance to biotic and abiotic stresses, physiological efficiency, quality of product and yield potential.

 Genetic diversity of wild and cultivated plants

Variation refers to any observable differences among living organisms; that is members of a species do not exhibit same characteristic even when grow in same environment. They differ from one another in one or more character these may include: height, form, nature of the leaf, mode of reproduction, floral morphology, fruit type, life cycle etc. This existence of wide diversity is useful in crop improvement as the potential’ source of genes’ besides the variation in the form, shape and flowering pattern provide aesthetics value to the landscape. Other plant parts are utilized for food & medicinal purposes. They also play a role in soil conservation (preventing soil erosion & nitrogen fixation). However some wild plants are weeds in crops & pasture and others are alternate hosts of common pest/disease in crops or have allelophatic effects on crops.

1.1  Wild plants

Currently the few remaining plants are endangered with deforestation, rapid urbanization and poor agricultural farming systems such as shifting cultivation & mono cropping. Therefore their conservation is essential in any future crop improvement. They can be found in the wild in the forest and the protected areas, arboretum’s, museums, road reserves, river banks and other uncultivated areas. They also act as gene banks still existing germplasm (in-situ conservation) and maintaining the ecological balance. Other uses include food, medicinal products and aesthetic values. Some of these crops can be serious weeds on crops/pastures, alternate host of common pest/disease in crops and poisonous to both human and livestock i.e. Datura stramonium.

Characteristics of wild plants

1. They shatter easily.
2. Non-uniformity in maturity
3. Have moderate disease/pest resistance,
4. Often have pronounced seed dormancy
5. Flowering early or late under stress conditions as a drought escape mechanism is common in crops of Solanaceae family.
6. Some plants produce many seeds.
7. They also have excellent varied dispersal mechanism(light in weight)and defence mechanism to enhance their survival even under extreme conditions these includes (explosive, wind and adhering to animal skin) and (leaf rolling, good cell stability, best adapted root systems presence of awns, needle leaves,) respectively.

1.2  Cultivated plants

Landraces are crops that have undergone a series of selection by the farmers who have grown over time; in each stage selection is done in order to obtain the best genotypes and lines based on their desired characters (quality, yields, tastes). Landrace consists of heterogeneous population (lines) which do not meet the requirements of DUS (Distinctness, Uniformity, Stability) and offers qualitative/technological advance compared to other registered varieties (VCU) (Value for Cultivation and Use); though they exhibit better resistance to biotic and abiotic stress.

Varieties/cultivars have been developed through crop improvement programs (breeding) do meet the requirements of DUS and VCU. Breeders identify heritable variation among the genotypes and concentrate on combining genes containing the trait of interest in a variety.

Characteristics of cultivated crops

          i.            Do not shatters easily making harvesting easier compared to wild plants.

        ii.            Uniformity in maturity.

      iii.            Good disease/pest resistance

      iv.            Seed dormancy is less pronounced and higher yields as well as good quality(aesthetic, baking quality).

        v.            Often have less pronounced seed dormancy.

      vi.            Do not flowering early or late under stress conditions.

    vii.            Some varieties have been bred to withstand different environmental stresses and agro-ecology.

4. GENE EXPRESSION

Reading assignment: read on the similarities & differences between DNA and RNA.

Genes are hereditary material/factors that are transmitted from parent to the offspring. This material is contained in chromosomes. They occupy specific positions in the chromosomes called loci and each locus contains Deoxyribonucleic acid (DNA).  Gene is a section of DNA on the chromosome made of bases along the strand. The DNA molecule a double helix strand made of a series of nucleotides. It is composed of a pentosesugar, phosphate and nitrogenous bases (Purines are Adenine & Guanine and Pyrimidines are Thymine, Cystosine). 

N/B: pairing as follows: A=T (double hydrogen bond) and   C&G form (triple hydrogen bond)

Basic structure of a nucleotide

 

P: phosphate

Roles of DNA

·         Stores genetic information in coded form

·         Transfer genetic information to daughter cells (replication)

·         Transfer genetic information to mRNA (transcription)

·         Expression of characters contained in genetic codes(translation)

Central dogma of life

Gene expression: This is the process by which information from a gene is used in the synthesis of a functional gene product. These gene products are often proteins (structural proteins, enzymes, hormones), but non-proteins coding genes such as ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA) genes the product is a functional RNA.

The main steps involved in gene expression are:

DNA transcription (occurs in the nucleus)

 Enzymes (DNA polymerase, helicase and nuclease)

         mRNA

DNA translation (occur in the cytoplasm)           

         tRNA

Formation of a polypeptide chain

N/B: Polypeptide chain is composed of a series of amino acids that later undergoes post-translation modification to form functional proteins (structural, hormones, enzymes).

The cells have capacity to control the structure and function of proteins a process called gene regulation. Gene regulation is the basis of cellular differentiation, morphogenesis and versatility & adaptability of organism by controlling the timing, location and the extent of expression. Gene expression is the most fundament level of which genotype give rise to phenotype which is the perceived visual expression of trait/character.

a)      DNA replication

For DNA replication to occur the following requirements must be in place; presence of the DNA template and free 3’-OH group. The initiation and continuation of DNA replication the DNA must be single strand. Presence of certain protein enzymes helps in breaking the double strand to be become a single strand through unwinding and separation into single strand. These enzymes include: DNA polymerase (exonuclease) which breaks the double strand into a single strand, DNA helicase which stimulates strand separation, DNA nuclease which add phosphate to the remaining phosphate – sugar backbone and Nuclease enzyme that removes wrong nucleotides from the daughter strand.

Refer to replication fork

b)     DNA transcription

It has been known that the information carried by DNA is used to specify the sequence of amino acids forming different kinds of the proteins i.e. enzymes, hormones and structural proteins.

This explains the one gene-one enzyme/polypeptide/protein hypothesis. Information contained in the DNA is transferred from the nucleus to the ribosomes in the cytoplasm where protein takes place through intermediary of RNA. N/B RNA consist of one polynucleotide chain. The DNA information is used to synthesis messenger RNA (mRNA) which moves from the nucleus to the cytoplasm. In the nucleus it attaches itself to ribosome’s where it directs the synthesis of specific protein. This process is similar to replication process except in this case we have ribo-nucleotides. The three base pairs would have 64 possible combinations.

N/B: 1. Thymine is replaced by Uracil.

         2. Genetic code refers to all codons specifying various amino acids.

         3. Codon is a triplet base sequence.

         4. There are 3stop codons (UAA, UGA &UAG).

c) DNA translation

This is the transfer of genetic information from mRNA to the sequence of amino acids in a protein. The coded message in mRNA is decoded and amino acids (aa) assembled into proteins. Free amino acids in the cytoplasm are brought to the ribosomes by the transfer RNA (tRNA). This is a short twisted molecule each specific for the 20 amino acids and at its centre tRNA has a sequence of three bases called anticodon that is complementary with mRNA.

Messenger RNA gets attached such that it passes through the centre of the ribosomes and is believed to move along the mRNA from one end to the other. As the ribosomes reads the codons the tRNA with complementary anticodon comes and attaches to the ribosome which then moves to the next and an appropriate tRNA comes in.  This process is repeated until the termination codon is reached meaning the polypeptide chain is complete or terminator codon is reached thus the chain is released from the ribosome to the cytoplasm where it undergoes modification to become active proteins while mRNA disintegrates into ribo-nucleotides.

The base sequence forms a codon which calls for specific amino acid (aa) for example TTT, CAA and CTA codes for lysine, valine and aspartic acid.  Proteins formed can be either structural or functional (enzyme/hormones) in the cell depending on the amino acid sequence.

The chart of the genetic codes and respective amino acid code

e.g.  TCT code for Serine

Refer to how ribosomes work

5. TISSUE CULTURE: THE MANIPULATION OF PLANT DEVELOPMENT

5.1. INTRODUCTION

Plant tissue culture is the in vitro (literally “under glass”) manipulation of plant cells and tissues, which is a keystone in the foundation of plant biotechnology. It is useful for plant propagation and the study of plant hormones, and is generally required to manipulate and regenerate transgenic plants. Whole plants can be regenerated in vitro using tissues, cells, or a single cell to form whole plants by culturing them on a nutrient medium in a sterile environment. Elite varieties can be clonally propagated, endangered plants can be conserved, virus-free plants can be produced by meristem culture, germplasm can be conserved, and secondary metabolites can be produced by cell culture. Besides this, tissue culture serves as an indispensable tool for transgenic plant production. For nearly any transformation system, an efficient regeneration protocol is imperative. This can be attributed to totipotency of plant cells and manipulation of the growth medium and hormones. Plant cells are unique in the sense that every cell has the potency to form whole new plantlike stem cells (stem cell production in mammals is located in time and space, and most mammalian cells cannot be converted to stem cells. However, having an understanding of each plant species and explant (donor tissue that is placed in culture) is essential to the development of an efficient regeneration system. The physiological stage of the explant plays a very important role in its response to tissue culture. For example, young explants generally respond better than do older ones.

This topic examines the history and uses of plant tissue culture and shows how it is integral to plant biotechnology, and presents the basic principles of media and hormones used in plant tissue culture, various culture types, and regeneration systems. Some people consider tissue culture as more of an art than science since the researcher must develop an eye for differentiating between good and bad (useful and non useful) cultures, which has often proved to be the difference between success and failure in plant biotechnology.

5.2. HISTORY

The history of plant tissue culture dates back at least to 1902, when Gottlieb Haberlandt, a German botanist, proposed that single plant cells could be cultured in vitro. He tried to culture leaf mesophyll cells, but did not have much success. Roger J. Gautheret, a French scientist, had encouraging results with culturing cambial tissues of carrot in 1934. The first plant growth hormone indoleacetic acid (IAA) was discovered in the mid-1930s by F. Kogl and his coworkers. In 1934 Professor Philip White successfully cultured tomato roots. In 1939 Gautheret successfully cultured carrot tissue. Both Gautheret and White were able to maintain the cultures for about 6 years by subculturing them on fresh media. These experiments demonstrated that cultures could be not only be initiated but also maintained over a long period of time. Later in 1955 Carlos Miller and Folke Skoog published their discovery of the hormone kinetin, a cytokinin. In 1962, Toshio Murashige and Skoog published the composition a plant tissue culture medium known as MS (named for the first letters of their last names) medium, which now is the most widely used medium for tissue culture. Murashige was a doctoral student in Professor Skoog’s lab, and they developed the now-famous MS medium working with tobacco tissue cultures. The formulation of MS medium took place while they were trying to discover new hormones from tobacco leaf extracts, which, when added to tissue cultures, enabled better growth. In a sense, their experiments could be deemed failures since they did not discover a new hormone. Nonetheless, they came up with a seemingly ideal medium for most plant tissue culture work that is used in practically every plant biotechnology laboratory around the world. This major breakthrough in the field of plant tissue culture has enabled nearly all the other breakthroughs cataloged in this book. MS medium seems to be ideal for many cultures since it has all the nutrients that plants require for growth and contains them in the proper relative ratios. The medium has high macronutrients, sufficient micronutrients, and iron in the slowly available chelated form. The success of tobacco culture using MS medium laid the foundation for future tissue culture work, and this has now become the medium of choice for most tissue culture work.

5.3. MEDIA AND CULTURE CONDITIONS

5.3.1. Basal Media

The success of tissue culture lies in the composition of the growth medium, hormones, and culture conditions such as temperature, pH, light, and humidity. The growth medium is a composition of essential minerals and vitamins that are necessary for a plant’s growth and development; everything, including sugar, which the plant needs to thrive all, must be in sterile or axenic conditions. The minerals consist of macronutrients such as nitrogen, potassium, phosphorus, calcium, magnesium, and sulfur, and micronutrients such as iron, manganese, zinc, boron, copper, molybdenum, and cobalt. Iron is seldom added directly to the medium; it is chelated with EDTA (ethylenediaminetetraacetic acid) so that it is more stable in culture and can be absorbed by plants over a wide pH range.

Note that EDTA is used in many foods as a preservative. If iron is not chelated with EDTA, it forms a precipitate, especially in alkaline pH. Vitamins are necessary for the healthy growth of plant cultures. The vitamins used are thiamine (vitamin B1), pyridoxine (B6), nicotinic acid (niacin), and thiamine. Other vitamins such as biotin, folic acid, ascorbic acid (vitamin C), and vitamin E (tocopherol) are sometimes added to media formulations.

Myoinositol, a sugar alcohol, is added to most plant culture media to improve the growth of cultures. In addition, plants require an external carbon source sugar since cultures grown in vitro rarely photosynthesize sufficiently to support the tissues’ carbon needs. Sometimes cultures are grown in the dark and do not photosynthesize at all. The most commonly used carbon source is sucrose. Other sources used are glucose, maltose, and sorbitol. The pH of the medium is important since it influences the uptake of various components of the medium as well as regulating a wide range of biochemical reactions occurring in plant tissue cultures (Owen et al. 1991). Most media are adjusted to a pH of 5.2–5.8. The acidic pH does not seem to negatively affect plant tissues but delays the growth of many potential contaminants. However, a higher pH is required for certain cultures. Cultures can be grown in either liquid or solid medium. The medium is most often solidified as it provides a support system for the explants and is easier to handle. Explant is the term denoting the starting plant parts used in tissue culture. Solidification is done using agar derived from seaweed or agar substitutes such as GelriteTM or PhytagelTM commercially available as a variety of gellan gums. These are much clearer than agar. Other than this membrane rafts or filter paper are also used for support on liquid medium.

A plethora of media formulations are available for plant tissue cultures other than MS (Murashige and Skoog 1962) are also used. McCown’s woody plant medium (WPM) has been widely used for tree tissue culture. Nitsch and Nitsch (1969) developed an culture. Knudson’s medium (Knudson 1946) was developed for orchid tissue culture and is also used for fern tissue culture.

With so many choices in media formulations, one might wonder about how to choose a medium to culture the species of interest. The choice of medium is typically determined empirically for optimal response of the plant species; explants used for culture and plant taxonomy are good starting points. For example, nearly all tissue cultures of plants in the Solanaceae (the nightshade family) use MS media. Recall that MS media was developed using tobacco, a member of this plant family. Many times a mix-and-match scheme of macro- and micronutrients from one medium and vitamins from another has also been successful. The composition of nutrients varies from medium to medium. For example, MS medium has higher macronutrients than does WPM, which is suitable for most plant species, but woody plants respond better in WPM than MS medium. It is important to select the right medium for culture according to how the plant empirically responds in tissue culture.

5.3.2. Growth Regulators

The basal medium (e.g., MS) is designed to keep plant tissues alive and thriving. Plant growth regulators or hormones are needed to manipulate the developmental program of tissues say, to make callus tissue proliferate, or produce roots from shoots. Growth regulators are the items most often manipulated as experimental factors to enhance tissue culture conditions. The most important growth regulators for tissue culture are auxins, cytokinins, and gibberellins. Both natural and synthetic auxins and cytokinins are used in tissue culture.

Auxins promote cell growth and root growth. The most commonly used auxins are IAA (indoleacetic acid), IBA (indolebutyric acid), NAA (naphthaleneacetic acid), and 2,4-D (2,4-dichlorophenoxyacetic acid). Cytokinins promote cell division and shoot growth. An auxin like compound TDZ (thidiazuron) has increased success rate of plant regeneration in many species. The most commonly used cytokinins are BAP (benzylaminopurine), zeatin, and kinetin. In addition to auxins and cytokinins, other hormones such as abscisic acid and jasmonic acid have also been used in plant cell culture. Other adjuvants (additional components that enhance growth) that have known to have a positive effect on morphogenesis are polyamines such as spermidine, spermine, and putrescene. By manipulating the amount and combination of growth hormones, regeneration of whole plants from small tissues is possible.

Another critical aspect in plant tissue cultures is the management of the gaseous hormone ethylene. When plants are grown in vitro in closed culture vessels, there is a buildup of ethylene, which is typically detrimental to the cultures. The addition of ethylene biosynthetic inhibitors such as silver nitrate, AVG (aminoethoxyvinylglycine), and silver thiosulphate have been shown to increase the formation of shoots.

Tissues are transferred to fresh media periodically every week to monthly, depending on the species and experiment. Without subculturing, tissues will deplete the media and often crowd each other, competing for decreasing resources.

 5.4. STERILE TECHNIQUE

5.4.1. Clean Equipment

Successful tissue culture requires the maintenance of a sterile environment. All tissue culture work is done in a laminar flow hood. The laminar flow hood filters air with a dust filter and a high-efficiency particulate air (HEPA) filter. It is important to keep the hood clean, which can be done by wiping it with 70% alcohol. The instruments used should also be dipped in 70% ethanol and sterilized using flame or glass beads. Hands should be disinfected with ethanol before handling cultures in order to avoid contamination. It is imperative to maintain axenic conditions throughout the life of cultures: from explants to the production of whole plants. Entire experiments have been lost because of an episode of fungal or bacterial contamination at any stage of culture. Especially problematic are fungal contaminants that are propagated by spores that might blow into a hood from an environmental source. Therefore, it is important to work away from the unsterile edge of a laminar flow hood. Culture rooms or chambers must be maintained as clean as possible to control any airborne contaminants.

5.4.2. Surface Sterilization of Explants

Plant tissues inherently have various bacteria and fungi on their surfaces. It is important that the explant be devoid of any surface contaminants prior to tissue culture since contaminants can grow in the culture medium, rendering the culture non-sterile. In addition, they compete with the plant tissue for nutrition, thus depriving the plant tissue of nutrients. Bacteria and especially fungi can rapidly overtake plant tissues and kill them. The surface sterilants chosen for an experiment typically depend on the type of explant and also plant species. Explants are commonly surface-sterilized using sodium hypochlorite (household bleach), ethanol, and fungicides when using field-grown tissues. The time of sterilization is dependent on the type of tissue; for example, leaf tissue will require a shorter sterilization time than will seeds with a tough seed coat. Wetting agents such as Tween added to the sterilant can improve surface contact with the tissue. Although surface contamination can be eliminated by sterilization, it is very difficult to remove contaminants that are present inside the explant that may show up at a later stage in culture. This internal contamination can be controlled to a certain extent by frequent transfer to fresh medium or by the use of a low concentration of antibiotics in the medium. Overexposing tissues to decontaminating chemicals can also kill tissues, so there is a balancing act between sterilizing explants and killing the explants themselves.

5.5. CULTURE CONDITIONS AND VESSELS

Cultures are grown in walk-in growth rooms or growth chambers. Humidity, light, and temperature have to be controlled for proper growth of cultures. A 16-h light photoperiod is optimal for tissue cultures, and a temperature of 22–258C is used in most laboratories. A light intensity of 25–50 mmol m22 s21 is typical for tissue cultures and is supplied by cool white fluorescent lamps. A relative humidity of 50–60% is maintained in the growth chambers. Some cultures are also incubated in the dark. Cultures can be grown in various kinds of vessels such as petri plates, test tubes, “Magenta boxes,” bottles, and flasks.

5.6. CULTURE TYPES AND THEIR USES

5.6.1. Callus Culture

Callus is an unorganized mass of cells that develops when cells are wounded and is very useful for many in vitro cultures. Callus is developed when the explant is cultured on media conducive to undifferentiated cell production usually the absence of organogenesis (organ production) can lead to callus proliferation. Stated another way, callus production often leads to organogenesis, but once callus begins to form organs, callus production is halted. Auxins and cytokinins both aid in the formation of most callus cells. Callus can be continuously proliferated using plant growth hormones or then directed to form organs or somatic embryos. Callus cultures can be transferred to a new medium for organogenesis or embryogenesis or maintained as callus in culture. Although callus has been induced for various reasons, one important application of callus is to induce somaclonal variation through which desired mutants can be selected.

5.6.1.1. Somaclonal Variation.

Plant cells undergo varying degrees of cytological and genetic changes during in vitro growth. Some of the changes are derived from preexisting aberrant cells in the explants used for culture. Others represent transient physiological and developmental disturbances caused by culture environments. Still others are a result of epigenetic changes, which can be relatively stable but are not transmitted to the progeny. Some variations are a result of specific genetic change or mutation and are transmitted to the progeny. Such genetically controlled variability is known as somaclonal variation.

Somaclonal variation serves as both a boon and a bane in tissue culture. It may hamper clonal propagation, but at the same time generate desirable somaclonal variants that can be selected for the development of novel cell lines. Induced somaclonal genetic variability of callus can give rise to genetically variable plantlets regenerated from callus and are of immense importance in the development and selection of various stress tolerant cell lines. Salt-tolerant (Ochatt et al. 1999), heavy-metal-tolerant (, disease-resistant (Jones 1990), and herbicide-resistant (Smith and Chaleff 1990) cell lines have been selected via somaclonal mutations using callus tissue.

5.6.2. Cell Suspension Culture

Lose friable callus can be broken down to small pieces and grown in a liquid medium to form cell suspension cultures. Cell suspensions can be maintained as batch cultures grown in flasks for long periods of time. Somatic embryos have been initiated from cell suspension cultures (Augustine and D’Souza 1997). Cell cultures have also been employed for the production of valuable secondary metabolites.

5.6.2.1. Production of Secondary Metabolites and Recombinant Proteins

Using Cell Culture. Plant cell cultures can be useful for the production of secondary metabolites and recombinant proteins. Secondary metabolites are chemical compounds that are not required by the plant for normal growth and development but are produced in the plant as “byproducts” of cell metabolisms. That is not to say that secondary metabolites serve no function to the plant; many do. Some are used for defense mechanism or for reproductive purposes such as color or smell. Some important secondary metabolites present in plants are flavonoids, alkaloids, steroids, tannins, and terpenes. Secondary metabolites have been produced using cell cultures in many plant species and have been reviewed by Rao and Ravishankar (2002). The process can be scaled up and automated using bioreactors for commercial production. Many strategies such as biotransformation, cell permeabilization, elicitation, and immobilization have been used to make cell suspension cultures more efficient in the production of secondary metabolites. Secondary metabolite production can be increased by metabolic engineering, in which enzymes in the pathway of a specific compound can be over expressed together, thereby increasing the production of a specific compound.

Transgenic plant cell cultures are gaining popularity in the large-scale production of recombinant proteins, thus making them integral parts of molecular farming. What makes molecular farming economically attractive is that production costs can potentially be much lower than those of traditional pharmaceutical production. Plant cell cultures are also advantageous for molecular farming because of high level of containment that they offer relative to whole, field-grown plants and the possibility of commercially producing recombinant proteins. Tobacco suspension culture is the most popular system so far; however, pharmaceutical proteins have been produced in soybean cells. So far, more than 20 pharmaceutical compounds have been produced in cell suspension cultures, which include antibodies, interleukins, erythropoietin, human granulocyte–macrophage colony-stimulating factor (hGM-CSF), and hepatitis B antigen.

5.6.3. Anther/Microspore Culture

The culture of anthers or isolated micropsores to produce haploid plants is known as anther culture or microspore culture. Microspore culture has developed into a powerful tool in plant breeding. Embryos can be produced via a callus phase or be a direct recapitulation of the developmental stages characteristic of zygotic embryos. It has been known that late uninucleate to early binucleate microspores are the best explants for embryogenesis. In this case, the somatic embryos develop into haploid plants. Doubled haploids can then be produced by chromosome-doubling techniques. Thus microspore culture enables the production of homozygous (at every locus) plants in a relatively short period as compared to conventional breeding techniques. These homozygous plants are useful tools in plant breeding and genetic studies. In addition, haploid embryos are used in mutant isolation, gene transfer, studies of storage product biochemistry, and physiological aspects of embryo maturation.

5.6.4. Protoplast Culture

Protoplasts contain all the components of a plant cell except for the cell wall. Using protoplasts, it is possible to regenerate whole plants from single cells and also develop somatic hybrids as described below. Cell walls can be removed from explant tissue mechanically or enzymatically; the latter is used most often. Enzymatic cell wall degradation was pioneered by Cocking (1960). Ever since then, protoplast production has been applied to various crop and tree species. Plant cell walls consist of cellulose, hemicellulose, and pectin, with lesser amounts of protein and lipid. Hence a mixture of enzymes is necessary for degrading the cell wall. The enzymes that are commonly used are cellulase and pectinase. Following enzyme treatment protoplasts are purified from cellular debris by filtering using a mesh and then flotation on either sucrose or ficoll. They are cultured in a high-osmoticum medium to avoid bursting. Protoplasts are cultured either on liquid or solid medium. Protoplasts embedded in an alginate matrix and then cultured on solid medium have better success rates of regeneration. The alginate provides cellular protection against mechanical stress and gradients in environmental conditions during the critical first few days of protoplast culture.

5.6.4.1. Somatic Hybridization.

Protoplast fusion and somatic hybridization techniques provide the opportunity for bypassing reproductive isolation barriers, thus facilitating gene flow between species. Fusion of protoplasts is accomplished by the use of PEG [poly(ethylene glycol)]. Protoplast fusion has helped in the development of somatic hybrids or cybrids (cytoplasmic hybrids). Protoplasts offer the possibility of efficient and direct gene transfer to plant cells. DNA uptake has been found to be easier in protoplasts than into intact plant cells. Although protoplasts seem to be a very attractive means for plant regeneration and gene transfer, they are very vulnerable to handling. One has to be very careful when manipulating protoplasts. They have to be cultured on a medium with a high osmoticum such as sucrose or mannitol; otherwise the protoplasts will burst open, which is why plant regeneration from protoplasts has proven to be difficult. Therefore, protoplasts are now used in cell culture studies mostly to study localization of proteins and transient transgene assays.

5.6.5. Embryo Culture

Embryo culture is a technique in which isolated embryos from immature ovules or seeds are cultured in vitro. This technique has been employed as a useful tool for direct regeneration in species where seeds are dormant, recalcitrant, or abort at early stages of development. Embryo culture also finds use in the production of interspecific hybrids between inviable crosses, whose seeds are traditionally condemned and discarded because of their inability to germinate. In plant breeding programs, embryo culture goes hand in hand with in vitro control of pollination and fertilization to ensure hybrid production. Besides this, immature embryos can be used to produce embryogenic callus and somatic embryos or direct somatic embryos.

5.6.6. Meristem Culture

In addition to being used as a tool for plant propagation, tissue culture is a tool for the production of pathogen-free plants. Using apical meristem tips, it is possible to produce disease-free plants. This technique is referred to as meristem culture, meristem tip culture, or shoot tip culture, depending on the actual explant that is used. Although it is possible to produce bacterium- or fungus-free plants, this method has more commonly been used in the elimination of viruses in many species. Apical meristems in plants are suitable explants for the production of virus-free plants since the infected plant’s meristems typically harbor titers that are either nearly or totally virus-free. Meristem culture in combination with thermotherapy has resulted in successful production of virus-free plants when meristem culture alone is not successful.

5.7. REGENERATION METHODS OF PLANTS IN CULTURE

In plant biotechnology, tissue culture is most important for the regeneration of transgenic plants from single transformed cells. It is safe to say that without tissue culture there would be no transgenic plants (although this situation is slowly changing nonetheless tissue culture is required to regenerate intact plants in most species).

5.7.1. Organogenesis

Organogenesis is the formation of organs: either shoot or root. Organogenesis in vitro depends on the balance of auxin and cytokinin and the ability of the tissue to respond to phytohormones during culture. Organogenesis takes place in three phases. In the first phase the cells become competent; next, they dedifferentiate. In the third phase, morphogenesis proceeds independently of the exogenous phytohormone.

Organogenesis in vitro can be of two types: direct and indirect.

5.7.1.1. Indirect Organogenesis.

Formation of organs indirectly via a callus phase is termed indirect organogenesis. Induction of plants using this technique does not ensure clonal fidelity, but it could be an ideal system for selecting somaclonal variants of desired characters and also for mass multiplication. Induction of plants via a callus phase has been used for the production of transgenic plants in which (1) the callus is transformed and plants regenerated or (2) the initial explant is transformed and callus and then shoots are developed from the explant.

5.7.1.2. Direct Organogenesis.

The production of direct buds or shoots from a tissue with no intervening callus stage is termed direct organogenesis. Plants have been propagated by direct organogenesis for improved multiplication rates, production of transgenic plants, and most importantly for clonal propagation. Typically, indirect organogenesis is more important for transgenic plant production.

5.7.1.2.1. Axillary Bud Induction/Multiple-bud Initiation.

This technique is the most common means of micropropagation since it ensures the production of uniform planting material without genetic variation. Axillary shoots are formed directly from preformed meristems at nodes, and chances of the organized shoot meristem undergoing mutation are relatively low. This technique is often referred to as multiple-bud induction.

Many economically important plants have been propagated using this method. Multiple bud initiation has been successful in crop plants but in only a few tree species such as Millingtonia hortensis. Multiple-bud initiation still remains a challenge in many tree species since many tree species are recalcitrant in tissue culture.

5.7.2. Somatic Embryogenesis

Somatic embryogenesis is a nonsexual developmental process that produces a bipolar embryo with a closed vascular system from somatic tissues of a plant. Somatic embryogenesis has become one of the most powerful techniques in plant tissue culture for mass clonal propagation. Somatic embryogenesis may occur directly or via a callus phase. Direct somatic embryogenesis is preferred for clonal propagation as there is less chance of introducing variation via somaclonal mutation. Indirect somatic embryogenesis is sometimes used in the selection of desired somaclonal variants and for the production of transgenic plants. Large-scale production of somatic embryos using bioreactors and synthetic seeds from somatic embryos has been successful.

Somatic embryos can be cryopreserved as synthetic seeds and germinated whenever necessary. One advantage of somatic embryogenesis is that somatic embryos can be directly germinated into viable plants without organogenesis; thus it mimics the natural germination process.

5.7.2.1. Synthetic Seeds.

Encapsulated somatic embryos are known as synthetic seeds. Somatic embryos are typically encapsulated in an alginate matrix, which serves as an artificial seed coat. The encapsulated somatic embryos can be germinated ex vitro or in vitro to form plantlets. Synthetic seeds have multiple advantages they are easy to handle, they can potentially be stored for a long time, and there is potential for scaleup and low cost of production. The prospects for automation of the whole production process are another advantage because the commercial application of somatic embryogenesis requires high-volume production. Synthetic seeds can be stored at 48^OC for shorter periods or cryopreserved in liquid nitrogen for long-term storage (Fang et al. 2004). Production of synthetic seeds and germination of these seeds to plantlets has been accomplished in sandalwood, coffee, bamboo, and many other plant species.

5.8. ROOTING OF SHOOTS

Efficient rooting of in vitro grown shoots is a prerequisite for the success of micropropagation. The success of acclimatization of a plantlet greatly depends on root system production. Rooting of trees and woody species is difficult as compared to herbaceous species. Rooting of shoots is achieved in vitro or ex vitro. Ex vitro (out of glass) rooting reduces the cost of production significantly. Ex vitro rooting is carried out by pre-treating the shoots with phenols or auxins and then directly planting them in soil under high humidity conditions. With this method, acclimation of the rooted shoots can be carried out simultaneously. In vitro rooting consists of rooting the plants in axenic conditions. Despite the cost factor, in vitro rooting is still a very common practice in many plant species because of its several advantages. Tissue culture conditions facilitate administration of auxins and other compounds, avoid microbial degradation of applied compounds, allow addition of inorganic nutrients and carbohydrates, and enable experiments with small, simple explants. Several factors are known to affect rooting. The most important factor is the action of endogenous and exogenous auxins. In many cases a pulse treatment with auxins for a short period has also been sufficient for root induction.

Phenolic compounds are known to have a stimulatory effect on rooting. Among the phenolic compounds, phloroglucinol, known as a root promoter, has a positive effect on rooting. Catechol, a strong reducing agent, has been reported to regulate IAA oxidation and thus affect rooting in plant tissue culture.

5.9. ACCLIMATION

Once plants are generated by tissue culture, they have to be transferred to the greenhouse or field. This requires that the plants be hardened-off before transfer to the field. During this acclimation process, plants are first transferred to a growth chamber or greenhouse and covered by domes to minimize the loss of water. Tissue culture conditions are at approximately 100% humidity, whereas relative humidity outside the vessels is typically much lower. In addition, the plants must be “weaned” off the rich media so they can grow as normal plants in soil. Once the plants are acclimatized under greenhouse conditions, they are ready for transfer to the field. Acclimation is a very important step in tissue culture since it is possible to lose plants if they are not properly hardened-off.

 5.10. CONCLUSIONS

Plant tissue culture is an essential tool in plant biotechnology that has enabled mass clonal propagation, production of secondary metabolites, preservation of germplasm, and production of virus-free plants. Moreover, it serves as an indispensable tool for regenerating transgenic plants. All this has been possible by manipulating plant tissues and various kinds of media developed by plant tissue culturists and by the use of plant hormones. It has been one of the very exciting discoveries for plant biologists and will continue to be most useful in the coming years.

6.                                               GENETIC TRANSFORMATION AND PRODUCTION OF TRANSGENIC PLANTS

Transgenic plants are those which carry additional stably integrated and expressed foreign gene(s), usually transferred from unrelated organisms. The whole process of introduction, integration and expression of foreign gene(s) in the host is called genetic transformation. Combined use of recombinant DNA technology, gene transfer methods and tissue culture techniques has lead to the efficient production of transgenics in a wide variety of crop plants. In fact, transgenesis has emerged as a novel tool for carrying out single gene breeding or transgenic breeding of crop plants. Unlike conventional plant breeding, here only the cloned gene(s) of agronomic importance are being introduced into plants without the co-transfer of other undesirable genes from the donor. The recipient genotype is least disturbed thereby setting aside the need for any backcross. This approach has the potential to serve as an effective means of removing certain specific defects of an otherwise well adapted cultivar which is not easily manageable through conventional breeding approaches.

It appears that through the process of transgenesis, any gene of interest can be transferred to the host to get a desired product. But the structure and mode of action of genes at molecular level reveals that the gene to be transformed does not constitute only the coding region specifying the sequence of amino acids in the protein product but includes other sequences in the DNA that determine the part of the plant, time and rate at which gene will be expressed: Such an information is embodied in the promoter region of the gene within which the enhancer sequences dictate the specific tissues and developmental stage for the expression of the gene. 'Promoters' for genes from non-plant sources usually express very poorly in plants and isolation of appropriate 'promoters' for transgenes becomes essential. A variety of 'promoters' are available some of which led to even increased expression of transgenes. Even after successful transformation, the cells or regenerated plants have to be screened to identify transgenic plants from amongst those which are genetically unaltered. Association of transgene with easily observable marker called reporter gene has to be established for effective screening and identification of transgenic plants. It thus is not only the addition of a structural gene but the transfer of a complete package of DNA sequences called gene construct encoding for structural gene, its normal expression and identification to identify transgenic plants. In this way transgenesis offers opportunities to develop new genotypes with desired levels of yield, quality, resistance, adaptability, etc. The transgenic plants can be produced even to serve as bioreactors, factories or designer plants for the production of speciality chemicals and pharmaceuticals enabling the adoption of molecular pharming.

6.1.   GENE TRANSFER METHODS

Gene transfer methods have broadened the available gene pool as the gene(s) may come to plants from unrelated plants, viruses, bacteria, fungi, insects, animals, human beings and even from chemical synthesis in the laboratory. The essential requirements to produce transgenic plants include the availability of appropriate gene construct, its transfer to plant "cells, integration and expression of transgenes and finally the characterization of transgenic plants. Depending upon the mode of transfer of foreign gene to plants two types of methods i.e. vector mediated and direct gene transfer are usually applied. In vector mediated approach the transgene is combined with a vector which takes it to the target cells for integration. In direct gene transfer, on the other hand, the gene is physically delivered to the target tissue. The systems available under each of these two categories are:

Vector Mediated

•     Agrobacterium tumefaciens '

•     Agrobacterium rhizogenes

•          Viral vectors

Direct Gene Transfer (DGT)

•     Physico-chemical uptake of DNA

•     Liposome encapsulation

•     Electroporation of protoplasts

•     Microinjection

•     DNA injection into intact plants

•     Incubation of seeds with DNA

•     Pollen tube pathway

•    Laser  micro beam

•    Electropporation  into  tissue  / embryos

•    Silicon  carbide fiber

•    Particle  bombardment

Fig 5. General scheme for genetic transformation and production of transgenic plants

6.1 Vector mediated gene transfer 

The vector mediated transformation is strongly linked to regeneration capabilities of the host plant. The target explants for vector mediated gene transfer usually include: protoplasts, suspension cells, callus cell clumps, cell layers, tissue slices or even whole organ sections. The cells to be used for transformation must be replicating DNA, which is available in wounded or dedifferentiated cells or protoplasts.

6.1.1    Agro bacterium-mediated Transformation

Agro bacterium is naturally occurring Bran-negative, soil bacteririum with two common species viz. A.tumefaciens and A.rhizogenes. These are known as natural genetic engineers for their ability to transform plants. In its natural environment, wild type Agrobacterium tumefaciens causes a crown gall disease. Genes for recognition of susceptible cells by the bacterium and its binding to these cells are located on bacterial chromosomes. However, the capability of bacterium to transfer DNA that is incorporated into plant's chromosome (transformation), resistance to antibiotics and pathogenicity are encoded on a plasmid. A plasmid is z segment of DNA which is separate from bacterial chromosome and can replicate independently of the chromosome. Wild type plasmid of A.tumefaciens is responsible for inducing tumour and hence termed as tumor inducing (Ti) plasmid. The Ti plasmid has two major regions of interest in transformation i.e. T-DNA and the vir region. The T-DNA region of the Ti plasmid of Agrobacterium is the part which is transferred to plant cell and incorporated into the nuclear genome of cells in an infected wound. The T-DNA part of plasmid carries genes which produce galls through regulation of phytohormones. Overproduction of phytohormones at the site of infection is responsible for the proliferation of wound cells into a gall (tumour) that can harbour a population of the bacteria. Transfer of the T-DNA is mediated by genes in another region of Ti-plasmid called vir (virulence) genes. So after infection of plants by bacteria, the single stranded T-DNA gets transferred to plant cells which actually express the plasmid genes to result in galls i.e. disease symptoms. If disease-inducing (phytohormone) genes are removed from T-DNA region of plastid i.e. plasmid is disarmed, the bacterium does not cause disease. On the other hand, if some other gene is placed in the T-DNA region, it is transferred to plant. So modified Ti plasmids are constructed that lack the undesirable tumour inducing genes but contain a foreign gene e.g. for Resistance to disease, and a closely linked selectable marker gene e.g. for antibiotic resistance within the T-DNA region. In this way any desired DNA-sequence i.e. gene can be transferred to plants through plasmids that becomes the basis of Agrobacterium mediated gene transfer. These properties of Agrobacterium DNA transfer system are invaluable for developing a powerful vector system for plant transformation. Any gene put in the T-DNA region of plasmid gets transferred to the plant genome and is inherently stable once in the plant genome because neither the border nor the virulence genes are transferred. Wild type Ti plasmid carrying phytohormone genes in the T-DNA region interferes with the plant regeneration process. Therefore, disarmed Ti plasmid (Ti plasmid without phytohormone genes) is generally used in the transformation process. Vector is maintained in the Agrobacterium either as cointegrating vector (where vector gets integrated into the Ti-plasmid) or as binary vector (where vector possesses autonomous replication). Transfer of genes through Agrobacterium is achieved either through co-culture of bacterium with plant cells or through application of Agrobacterium to wounded plant organs.

Fig 6. Structure of Ti plasmid

Method of transformation. intact  plants  and  seedling  explants  such as  cotyledons, hypocotylas, roots, calli and  protoplasts can be  used for  co-cultivation with  agrobacterium cells  containing  recombinant  plasmids. However  , leaf  disc  method  has been  widely  used  where surface  sterilized  leaf  disc  are infected  with  the  appropriate  strain  of agrobacterium  carrying  the   vector of choice  and  co-cultured on regeneration medium for  two  or  three days  .  during   this  time , the  virulences genes in   the bacteria are  included the , bacteria bind to the plant cells  around  the wounded site and  the gene trasfer  occurs. The leaf discs   are  then transferred to  regeneration/ selection medium which contains 500 ug/ml carbenicillin to hull the Agrobacteria and the appropriate antibiotic, usually kanamycin, to inhibit the growth of untransformed plant cells. During next 4-5 weeks, 211e transformed shoots are obtained which are rooted and transferred to soil. Transgenic plants regenerated frown various tissues are called T_o oiants whereas their subsequent generations are called as T, T₂, T, e.t.c

When Agrobacterium rhizogenes infects plants, adventitious roots rather than tumor are formed at the site of infection. This is mediated by Ri (root inducing) plasmid. Vectors such as pRiAu which are based o l Ri plasmid have been developed. The Ri vectors are particularly useful for studying nodulation and manipulating root cultures for secondary metabolite and Vascular Arbuscular Mycorhiza (VAM) production.

Agroboeterium and host range. Agrobacterium mediated transformation has been a method of choice in dicotyledonous plant species where plant regeneration systems are well established. The host range of this pathogen includes about 600 of gymnosperms and dicotyledonous angiosperms. Besides, transformation success has also been achieved in some monocots like Asparagus officinalis, chlorophytum, Narcissus. It was believed that monocots lack wound response i.e. factors that are required to initiate Agrobacteriain infection via induction of `vir' genes. Schafer et al. (1987) achieved success in transforming another monocot, yam (Dioscorea bulbifei^-a) by treating Agrobacteria with wound exudate (phenolic compounds) from potato tubers. Likewise, the use of aeetosyringone (synthetic phenolic compound) either during bacterial growth or during co-cultivation has been found to be beneficial in the transformation of other monocots. There are several reports on successful transformation of rice using Agrobacterium. This method has also been extended to barley, wheat, maize and sugarcane.

6.1.2    Viral vectors

On account of their ability to cause systemic infections in the plants, the viruses are being investigated as vectors for gene transfer in plants. Genetic engineering of the genomes of DNA and RNA viruses has been accomplished with the introduction of foreign DNA sequences. The foreign genes replace a part of the viral genome and create a defective viral particle that can infect the target plant only in the presence of a helper virus. The most promising viruses belong to two groups having DNA genome viz. Cauliflower Mosaic Virus (CaMV) and gemini virus. But viral vectors have not been developed to a stage where these can be routinely used for plant transformation.

6.2 Direct Gene Transfer Methods

The problems associated with the species specificity and the inability of Agrobacterium to transfer multiple genes has been circumvented through direct gene transfer methods without the involvement of biological agents like bacteria and viruses. Here the DNA to be inserted in the target cells is delivered either through direct uptake by cell or through certain physical and chemical processes. A number of methods have been developed which include chemical methods, electroporation, particle gun delivery, lipofection, microinjection, macroinjection, pollen transformation, delivery via growing pollen tubes, laser induced or fibre-mediated gene transfers. Some of these like lipofection and electroporation involve delivery of DNA to protoplasts and thus require regeneration of plants from transformed protoplasts. The techniques like micro- or macroinjection,on the other hand, can be applied to a number of explants.

6.2.1             Physico-chemical Uptake of DNA

It is based on the ability of the protoplast to uptake the foreign DNA from the surrounding solution. An isolated plasmid DNA (vector) is mixed with the protoplasts in the presence of polyethylene glycol (PEG), polyvinyl alcohol and calcium phosphate which enhance the uptake of DNA by protoplasts. After 15-20 minutes of incubation, the protoplasts are cultured in the presence of appropriate selective agent. Protoplasts are regenerated and putative transgenic plants are further characterized for confirmation (Paszkowski et al.'1984). This method, however, depends on the plant regeneration ability of the protoplasts and has been successfully used to produce transgenic plants in brassica, strawberry, lettuce, rice, wheat and maize.

 6.2.2 Liposome Encapsulation

Liposomes are small lipid bags enclosing large number of plasmids. The procedure of liposome encapsulation was developed to protect the foreign DNA during the transfer process (Deshayes et al. 1985). The DNA enclosed in the lipid vesicles when mixed with protoplasts under appropriate conditions, penetrates into the protoplasts where lipase activity of the protoplast dissolves the lipid vesicles and DNA gets released for integration into the host genome. This method has not been commonly used as it is difficult to construct the lipid vesicles-and the success depends upon the protoplast regeneration.

6.2.3 Electroporation of Protoplasts

Electroporation involves the creation of pores in the protoplast membrane using electrical impulses of high field strength. Reversible breakdown of the membrane allows the entrance of foreign DNA into cytoplasm. Generally, protoplasts are used since they have exposed plasma membrane. Protoplasts are suspended in buffered saline solution containing plasmid DNA in a cuvette and an electrical pulse is applied across two platinum electrodes in the cuvette. The protoplasts are then cultured to regenerate the plants (Fromm et al. 1985). Electroporation has been successfully used for obtaining transgenics in tobacco, maize and rice (Joresbo and Brunsted, 1991).

6.2.4. Microinjection

Although it has been extensively used for animal cells but microinjection of DNA into plant cells has not been very successful. It is largely because of difficulties in getting the protoplasts immobilized and in injecting DNA into the protoplast without damaging the tonoplast which surrounds the plant cell vacuole. Tonoplast damage leads to the release of several kinds of toxic substances from the vacuoles into the cytoplasm. Crossway et al. (1986) developed the "holding pipette" technique to microinject tobacco protoplasts to overcome some of these problems. Protoplasts are held onto a 5-10 um pipette by gentle suction. Foreign DNA (about 2 picolitres) is injected into nucleus of the protoplast using 0.2 um diameter injection pipette. After microinjection the protoplasts are cultured to obtain entire plants. The process of microinjection is time consuming and technically difficult but it raises the possibility of microinjecting a variety of materials such as chromosomes and even chloroplasts and mitochondria can also be transferred by microinjection. It has been successfully used in tobacco, alfalfa and Brassica spp.

6.2.5        DNA Injection into Intact Plants (Macroinjection)

Transgenic plants have been reported through direct DNA injection in the intact plants of rye (de La Pena et al. 1987). Macroinjection involves injection of a relatively large volume of exogenous DNA solution into inflorescence using a syringe. An aqueous solution of DNA was introduced into developing floral tillers 14 days prior to meibsis. Transformed seeds were obtained from these injected tillers after cross pollination with other injected tillers. However, the mechanism by which DNA entered the zygotic tissue is yet unknown.

6.2.6 Incubation of Seeds with DNA

In some of the earlier experiments transformations were observed when germinating seeds were put into the DNA solution. Dry seeds without seed coats also take up DNA when imbibed in a DNA solution.

6.2.7 Pollen Tube Pathway

Transgenic plants have been reported in rice by the percolation of DNA solution through the pollen tube which eventually resulted in the formation of transformed seeds (Luo and Wu, 1988). The stigmas were cut after pollination exposing the pollen tubes and the DNA was introduced onto the cut surface that presumably diffused through the germinating pollen tube into the ovule. The mechanism of DNA transfer into zygote through this method is not yet established.

6.2.8     Laser Microbeam

A method of introducing DNA into plant cell with a c/V-laser microbeam has been described by Weber et al. (1988). Small pores in the membrane are created by laser microbeam. The DNA from the surrounding solution then enters the cell through these small pores to cause transformation.

6.2.9     Electroporation into Tissues/embryos

A procedure has been developed to deliver DNA by electroporation into intact cultured cells, immature and mature embryos. This procedure has been successfully used to obtain transgenic plants by delivering DNA into enzymatically or mechanically wounded immature embryos, embryogenic callus and bisected mature embryos of rice. Concerted efforts are required to improve the transfor­mation frequency of this inexpensive procedure.

6.2.10 Silicon Carbide Fibre

The DNA can be delivered into intact plant cells i.e. cytoplasm and nucleus by a simple and inexpensive method which involves the vortexing of suspension culture cells in a medium containing silicon carbide Fibers ,(0.6 um x 10-80 um) and plasmid DNA.

6.2.11 Particle Acceleration

In its basic concept the process of particle acceleration or biolistics involves acceleration of DNA into cells with sufficient force such that a part of it gets integrated into DNA of the target cells. The process of transformation employs foreign DNA coated minute 0.2-0.7 um gold or Kingston particles to deliver into target plant cells. Two procedures have been used to accelerate minute particles i.e. by using pressurized helium gas or by electrostatic energy released by a droplet of water exposed to a high voltage. The original equipment used a gunpowder charge behind a packed cartridge to accelerate the particle that gave it the name of ‘particle gun'. 'Particle gun' accelerated particles penetrate even deep into the tissue (Klein et al. 1987; Sanford, 1988). This method is being widely used because of its ability to deliver foreign DNA into regenerable cells, tissues or organs irrespective of the monocots or dicots. Because of the physical nature of the process, there is no biological limitation to the actual DNA delivery that makes it genotype independent. It can be used to transform shoot apical meristems, leaf blades, pollen, cultured cells, root and shoot sections. The transgenic plants have been recovered in several important crops including barley, cotton, maize, rice, soybean, sugarcane, sunflower and wheat by using more .regenerable tissues/organs such as embryogenic calli; immature embryos and shoot meristems (Christou, 1994).' Potrykus (1993) while emphasizing the advantages of this method expressed that it allows the transport of genes into many cells at nearly any desired position in an experimental system without too much manual labour.

6.3       TRANSIENT AND STABLE GENE EXPRESSION

After successful introduction of foreign gene it does not always integrate into the genome of the recipient cells. The introduced gene may, therefore, express in a transient fashion. This activity, of course, declines over time and eventually disappears and is not desirable if interest lies in the production of transgenic plants. However, the transient assays, 24-48 hours after DNA delivery of the reporter genes help in the rapid evaluation of transformation protocols and the suitability of the plasmid constructs. In addition such assays have proved to be most useful to obtain basic information about gene regulation and function. When the introduced gene gets stably integrated with recipient genome and its subsequent expression is called as stable gene expression

6.4      GENETIC MARKERS IN TRANSFORMATION

In the process of transformation the DNA is delivered only to a fraction of cells involved in the experiment and gets stably integrated into the nuclear genome of only fraction of cells that receive DNA and finally the introduced gene(s) are expressed in still fewer cells. Therefore, preferential selection of rare transformed cells from a large population of non-transformed protoplasts/cells is critical step affecting success rate of the transformation programme. The transformed cells or plants are usually identified indirectly through marker genes closely linked to the gene of interest. In the absence of selection, the non-transformed cells overgrow the transformed ones. A selective advantage can be given to the transfo­rmed cells through the introduction of a gene that will allow them to grow under conditions which inhibit growth of non-transformed cells. The transformed celss can also be identified when expression of trangene is directly observable i.e. gene itself is detectable e.g for herbicide resistance. All the non-transformed cells are killed by herbicide leaving only transformed ones. In other cases a gene called reporter gene can be added as a marker for the identification of transformed cells. Such markers are of two types’ i.e. screenable (scorable) markers and selectable markers. The screenable markers help in screening for transformed cells through the expression of specific enzymes to produce distinctive phenotype. Some of commonly used detectable enzyme producing genes are nos, lux, cat, gus and m₂fpu(table1). No selection pressure is applied on cells or regenerated shoots and only small pieces of cut plant tissues are observed for expression of gene marker. As an instance gus gene produces enzyme that acts on the substrate within cells and is converted into a blue precipitate visible to naked eye. With green fluorescent protein gene the transgenic tissues gives green fluorescence under UV illumination. The selectable markers on the other hand, are the genes which confer resistance to various compounds like herbicides and antibiotics that usually are toxic to normal plant cells so that only those cells having these markers survive under selective conditions. The selectable markers thus allow selective multiplication of transformed cells by killing the non transformed cells, whereas scorable markers only enable the identification of the transformed cells without addition of any toxic compound. The commonly used screenable and selectable markers have been listed in table 1 blow.

6.5    CONFIRMATION OF TRANSFORMATION

Plants are obtained in several ways transformation. Several independent

(i) Phenotypic Assay (ii) Enzymatic assay (iv) Southern blot analysis (vi) Progeny analysis

6.5.1Phenotypic Assay

The easiest and most obvious indication of effective transformation is when the transgenic individual displays the anticipated phenotypic effect. A plant is transformed if it grows in the presence of elevated concentration of selective compounds including antibiotics and herbicides. Transgenics exhibit profuse hairy roots, lack of geotropism and plants with wrinkled leaves when a wild type Agrobacterium rhizogenes is used.

6.5.2  Enzyme Assays

Enzyme assay of a genetic marker (nos, cat) is done to check the expression of the foreign DNA in the transformed tissue. Different genes express at different levels in different tissues but enzyme assays are generally done using rapidly expanding tissues.

6.5.3  PCR Analysis

The polymerase chain reaction (PCR) amplifies DNA sequences between defined synthetic primers. A set of primers (forward primer and reverse primer) which is specific for the transgene is used to selectively amplify the transgene sequence from the total genomic DNA isolated from putative transgenic tissue/plant. The PCR product can indicate the presence or absence of the transgene but PCR usually amplifies a part of the gene and not the whole cassette. It is good for preliminary screening but due to DNA contamination it may lead to false positives. The PCR fails to tell anything about the transgene copy number, integration sites, intactness of the cassette and the expression level of the transgene.

6.5.4 Southern Blot Analysis

Southern blot hybridization is an efficient method for transferring DNA from agarose gels onto membranes prior to hybridization (using either radioactive or nonradioactive probes). It is very sensitive technique which is used to detect the transgene in the genomic DNA even without any amplification. Southern analysis tells about the (1) stable integration of transgene into the genome, (2) copy number of the transgene and (3) number of integration sites. However, it does not tell anything about the expression of transgene.

6.5.5 Western Blot Analysis

This analysis involves the detection of proteins produced by transgene in transgenic plant and is a reliable technique for analyzing the expression of transgenes. The level of gene expression is estimated by calculating the amount of protein produced by the transgene and its proportion in the total soluble plant protein.

6.5.6 Progeny Analysis

In annual plants heritability of introduced gene can be easily deteiniined by selfmg orbackcrossing of the putative transgenics. Transgene segregation can be studied by analysing 7\ and subsequent generations.

6.6 GENE SILENCING IN TRANSGENIC PLANTS

The stably integrated transgenes are usually inherited in Mendelian fashion but under many situations the new phenotypes created through transgenesis exhibit loss of newly acquired traits after propagation. Such a loss of expression does not suggest the loss of the transgene but indicates its inactivation. Major causes of gene inactivation include: methylation of DNA sequences, environmental factors, co-suppression caused by the introduction of multiple copies of the foreign gene and chromoplast dysfunction by transinactivation. Gene silencing process i.e. suppressed expression of transgenes may be operative at transcriptional or post-transcriptional levels. Transcriptional silencing usually occurs due to methylation of promoter region. The integration of multiple copies of transgene during transformation causes hyper-methylation. Integration of transgene may often occur in certain hypermethylated regions of chromosome that causes reduced or variable expression of transgene. Post-transcriptional mechanisms are also of
above type where methylation may occur in coding region of gene but does
not directly affect transcription. A stable and optimum level of expression can be attained through planned breeding i.e. new population can be synthesized by crossing different transgenic lines and screening to identify lines with enhanced and stable expression.

7.         ENGINEERING CROPS FOR USEFUL AGRONOMIC TRAITS

Remarkable achievements have been made in the production, ' characterization and field evaluation of transgenic plants in several field, fruit and forest plant species. However, the major interest has been in the introduction of cloned gene(s) into the commercial cultivars for their incremental improvement. Using different gene transfer methods and strategies, transgenics have been developed in several crops carrying useful agronomic traits (Dale et al. 1993; Shah et al. 1995; Hansen and Wright, 1999). The research and development efforts on genetic transformation of crop plants are receiving priority in most of the important crop plants. The latest figures on area under transgenic plants reveal that over 90 per cent of area under transgenic plants relates to three crops viz. soybean, maize and cotton. In all these three crops the area has more than doubled from 1997 to 1998.During the year 2000, the area under transgenic crops has been increased to 44.2 million hectares spread over 13 countries of the world. There has been an apparent increase in emphasis on the release of transgenic varieties in soybean which alone contributes over half of the total acreage under genetically transformed plants. The available statistics (Table 26.4) further highlight the importance of transgenic plants in breeding for resistance to herbicides and insects. In general main achievements in transgenesis include production of transgenics for insect resistance; herbicide resistance; disease resistance; alteration of nutritional quality and biochemical production.

INSECT RESISTANCE

Detoxification

Transfer of gene whose enzyme product detoxifies the herbicide In this approach the introduced gene produces an enzyme which degrades the herbicide sprayed on the plant. Introduction of bar gene cloned from bacteria, Streptomyces hygroscopicus, into plants makes them resistant to herbicides based on phosphinothricin (ppt). The bar gene produces an enzyme' phosphinothricin acetyl transferase (PAT) which degrades phosphinothricin into a non-toxic acetylated form. Plants engineered with: Jar genes were found to grow in 'ppt' at levels 4-10 times higher than normal field application. Likewise, bxn gene of Klebsiella ozaenae which produces nitrilase enzyme imparts resistance to plants against herbicide hromoxynil. A transgenic cotton variety "BXN cotton" resistant to hromoxynil has been released in USA. Other genes that have been used for herbicide resistance include tfdA for 2,4-D tolerance and GST gene for Atrazine tolerance.

Target Modification

Transfer of gene whose enzyme product becomes insensitive to herbicide In this approach a mutated gene is introduced and produces modified enzyme in the plant that is not recognized by the herbicide as a consequence of which it cannot kill the plant. For instance a mutant aroA gene from bacteria Salmonella typhimurium has been used for developing tolerance to herbicide, glyphosate. The target site of glyphosate is a chloroplast enzyme 5-enol pyruvylshikimic acid 3-phospha'e synthase (EPSPS). Introduction of mutant aroA gene produces modified- EPSPS, not recognizable to glyphosate. 'Roundup Ready' soybean and 'Roundup Ready' cotton varieties resistant to glyphosate have been released. Likewise, sulphonylurea and imidazolinone herbicides inhibit acetolactate synthase (ALS) chloroplast protein. Tolerance to these herbicides has been achieved by engineering the expression of the mutant gene 'ALS' derived from plant.

ENGINEERING DISEASE RESISTANCE

Virus Resistance

 Genetic engineering of virus resistant plants has exploited new genes derived from viruses themselves in a concept referred to as 'pathogen-derived-resistance' (PDR) through the processes of:

(i)       Coat-protein-mediated resistance (CP-MR): This is achieved by transforming plants with the viral genes which encode coat proteins. The accumulation of this protein in uninfected cells results in effective resistance by uncoating the virus particles before translation and replication. Introduction of viral coat-protein gene into the plant makes it resistant to virus from which the gene for the coat-protein was derived. It was first demonstrated for TMV in tobacco. Subsequently virus resistant trangenics have been developed in tomato, melon, rice, papaya, potato and sugarbeet. A variety of yellow squash called Freedom II possessing virus resistance has been released in U.S.A. Several, CP-MR varieties of potato, cucumber and tomato are under field evaluation.

(ii) Satellite RNAs mediated resistance: Some RNA viruses have small RNA molecules called satellite RNAs which show little, if any, sequence homologies with the virus to which they are associated, but are replicated by the virus polymerases and appear to affect the severity of infection produced by the virus. The presence of sat-RNA leads to reduction in severity of disease symptoms and thus has been used to develop resistance against specific viruses. It has been demonstrated that engineering cucumber using cucumber mosaic virus (CMV) satellite RNA leads to transgenics resistant to CMV. Similarly transgenic tobacco plants have been developed that produce species of sat-RNA that reduce the severity of disease.

(Hi) Antisense mediated protection: It is now established that gene expression can be controlled by antisense RNA. In a normal gene only one strand of DNA called antisense strand transcribes whose base sequences are complementary to the sequences of other strand i.e. sense strand. In antisense RNA technology the antisense gene is produced by inverting the orientation of coding region in relation to promoter. So the natural sense strand of gene becomes antisense and is transcribed. The presence of endogenous gene and antisense gene produce antisense and sense RNAs which are complementary to each other and thus pair as double-stranded RNA so that mRNA is not available for translocation leading to silencing of normal gene (Fig. 26.3). So the production of antisense-RNA for a given gene results in production of two different but complementary RNAs which hybridize to inhibit translation and thus cause post-transcriptional down-regulation of gene activity. It has been proposed that antisense RNA technology can also play a role in cross protection. The cDNAs representing viral RNA genomes have been cloned in an antisense orientation to a promoter and transferred to plants. The introduction of antisense viral genes into plants will therefore inhibit coat protein production and thus will limit the production of new viral particles in infected cells. This approach has been effective against TMV although the protection was not as effective as with coat protein gene. The antisense technology in fact was originally developed to regulate fruit ripening by down-regulation of fruit ripening genes.

Fig7. Anti sense RNA technology

Fungal Resistance

Though genetic engineering for fungal resistance has been limited but several new advances in this area now present an optimistic outlook.

(i) Antifungal protein-mediated resistance: The two genes coding for chitinase and glucanase are receiving attention for the production of disease resistant transgenic plants. These two genes are anticipated to attack the cell walls of invading fungi to render them more susceptible to natural plant defences. Introduction of chitinase gene in tobacco and rice has been shown to enhance the fungal resistance in plants. Chitinase. enzyme degrades the major constituents of the fungal cell wall (chitin and 6-1,3-glucan). Co-expression of chitinase and glucanase genes in tobacco and tomato plants confers higher level of resistance than either gene alone. The use of genes for ribosome inactivating proteins (RIP) along with chitinase gene has also shown synergistic effects. A radish gene encoding antifungal protein 2(Rs-AFP2) was expressed in transgenic tobacco and resistance to Alternaria longipes was observed (Baron and Zambryski, 1995)

(ii) Antifungal-compound mediated resistance: The low molecular weight compounds such as phytoalexins possess antimicrobial properties and have been postulated to play an important role in plant resistance to fungal and bacterial pathogens. Expression of a stilbene synthase gene from grapevine in tobacco resulted in the production of new phytoalexin (resveratrol) and enhanced resistance to infection by Botrytis cinerea and blast resistance in rice (Starklorenzen et al. 1987). Active oxygen species (AOS) including hydrogen peroxide also play important role in plant defense responses to pathogen infection. Transgenic potato plants expressing an H₂0₂ generating fungal gene for glucose oxidase were found to have elevated levels of H₂0₂ and enhanced levels of resistance both to fungal and bacterial pathogens particularly to Verticillium wilt. The introduction of H₂0₂ gene has also improved fungal resistance in rice (Anthony et al. 1997).

Bacterial Resistance

Genetic engineering for resistance to bacteria has met with relatively little success. The expression of a bacteriophage T₄ lysozyme in transgenic potato tubers led to increased resistance to Erwinia carotovora. Besides^ the expression of barley a-thionin gene significantly enhanced the resistance of transgenic tobacco to bacteria Pseudomonas syringae. Advances in the cloning of several new bacterial resistance genes such as the A rabidops is Rps2 gene, tomato Cf9 and tomato Pto gene may provide better understanding in the area of plant bacterial interactions.

ENGINEERING FOR MALE STERILITY

Dominant nuclear male sterility and restorer genes have been successfully engineered into plants. This system utilizes the tapetal specific transcriptional activity of the tobacco TA29 gene and an RNase (barnase)/ RNase-'inhibitor (barstar) from bacterium, Bacillus amyloliquefaciens. The introduction and expression of male sterility gene construct [TA29, tapetum specific promoter-barnase gene for cytotoxic protein] destroys tapetal cells, inhibits pollen formation and causes male sterility. The introduction and expression of restorer gene construct [TA29, tapetum specific promoter-barstar (restorer gene)] into another plant leads to the development of restorer line. Barstar is the specific inhibitor of barnase; inhibition involves the formation of a stable, noncovalent, one to one complex of the two proteins. Expression of barstar in the male parent has no effect on tapetal development and the plants are male fertile. The male sterility due to barnase is dominant and it is maintained by crossing male sterile (barnase/-) with normal non-transformed male fertile parental line (-/-)i.e. without barnase). But the progeny of a cross of sterile (barnase/-)-and normal fertile plants (-/-) will produce 50 percent sterile (barnase/-) and 50 percent fertile (-/-) plants. The fertile plants shall have to be removed before flowering. The identification of fertile plants is achieved by linking barnase gene with bar gene that confers resistance to herbicide phosphinothricin, as a gene construct barnase-bar. So all the male sterile plants (barnase-bar) are resistant to herbicide whereas male fertile (-/-) are susceptible and thus killed by herbicide spray. When male sterile plants are crossed with restorer male fertile plants (barstar), the resulting hybrid, contains and expresses both genes. The two proteins form an inactive complex, tapetal development is normal and the hybrid is fully fertile. The genetically engineered lines of oilseed rape and maize i.e. NMS (TA 29-baraase) and Rf (TA29-barstar) are being evaluated for commercial hybrid seed production. Besides barnase/barstar there are several other genes such as arg E, BcPI gene causing sterility and restoration but the major success has met only with the barnase-barstar system.

ENGINEERING FOR FOOD PROCESSING/QUALITY

Ripening of many fruits and vegetables is governed by the ethylene production, the regulation of which helps in controlling over-ripening that increases shelf life. By using antisense-RNA technique the antisense gene which inhibits the expression of polygalacturonase (PG) reduces softening and prevents over ripening of tomato. Fruits of plants with this antisense gene have very little or no PG activity and soften only slowly. The Flavr Savr™ is genetically engineered tomato containing antisense construct of encoding PG to reduce its activity and thus possess greater shelf life. The phaseolin gene has been transferred into tobacco and chicken ovalbumin gene in alfalfa to obtain better protein quality.

ENGINEERING FOR ABIOTIC STRESS TOLERANCE

Cloned genes have been transferred to produce transgenics even for resistance to abiotic stresses. For instance, for frost protection an antifreeze protein gene from fish has been transferred into tomato and tobacco. Likewise, a gene coding for glycerol-3-phosphate acyltransferase from Arabidopsis has been transferred to tobacco for enhancing cold tolerance. The genetic control of environmental stresses is usually complex involving many genes. Though not much success has yet been reported in engineering plants for abiotic stresses but recent developments in quantitative trait loci (QTL) mapping may provide new avenues to identify, characterize and transfer useful DNA segments from wild species which are known to be hardy for survival under adverse environmental conditions.

           

Table 2. Important transgenes/transgene products being used for engineering crops with useful agronomic traits.

8. ISSUES OF GENETICALLY MODIFIED FOODS

Environmental activists, religious organizations, public interest groups, professional associations and other scientists and government officials have all raised concerns about GM foods, and criticized agribusiness for pursuing profit without concern for potential hazards. It seems that everyone has a strong opinion about GM foods. Even the Vatican and the Prince of Wales have expressed their opinions. Most concerns about GM foods fall into three categories: environmental hazards, human health risks, and economic concerns.

Environmental hazards

 Unintended harm to other organisms: Last year a laboratory study showed that pollen from B.t. corn caused high mortality rates in monarch butterfly caterpillars. Monarch caterpillars consume milkweed plants, not corn, but the fear is that if pollen from B.t. corn is blown by the wind onto milkweed plants in neighboring fields, the caterpillars could eat the pollen and perish. Although the Nature study was not conducted under natural field conditions, the results seemed to support this viewpoint. Unfortunately, B.t. toxins kill many species of insect larvae indiscriminately; it is not possible to design a B.t. toxin that would only kill crop-damaging pests and remain harmless to all other insects. This study is being reexamined by the USDA, the U.S. Environmental Protection Agency (EPA) and other non-government research groups, and preliminary data from new studies suggests that the original study may have been flawed. This topic is the subject of acrimonious debate, and both sides of the argument are defending their data vigorously. Currently, there is no agreement about the results of these studies, and the potential risk of harm to non-target organisms will need to be evaluated further.
 Reduced effectiveness of pesticides: Just as some populations of mosquitoes developed resistance to the now-banned pesticide DDT, many people are concerned that insects will become resistant to B.t. or other crops that have been genetically-modified to produce their own pesticides.
 Gene transfer to non-target species: Another concern is that crop plants engineered for herbicide tolerance and weeds will cross-breed, resulting in the transfer of the herbicide resistance genes from the crops into the weeds. These "superweeds" would then be herbicide tolerant as well. Other introduced genes may cross over into non-modified crops planted next to GM crops. The possibility of interbreeding is shown by the defense of farmers against lawsuits filed by Monsanto. The company has filed patent infringement lawsuits against farmers who may have harvested GM crops. Monsanto claims that the farmers obtained Monsanto-licensed GM seeds from an unknown source and did not pay royalties to Monsanto. The farmers claim that their unmodified crops were cross-pollinated from someone else's GM crops planted a field or two away. More investigation is needed to resolve this issue.

 There are several possible solutions to the three problems mentioned above. Genes are exchanged between plants via pollen. Two ways to ensure that non-target species will not receive introduced genes from GM plants are to create GM plants that are male sterile (do not produce pollen) or to modify the GM plant so that the pollen does not contain the introduced gene. Cross-pollination would not occur, and if harmless insects such as monarch caterpillars were to eat pollen from GM plants, the caterpillars would survive.

 Another possible solution is to create buffer zones around fields of GM crops. For example, non-GM corn would be planted to surround a field of B.t. GM corn, and the non-GM corn would not be harvested. Beneficial or harmless insects would have a refuge in the non-GM corn, and insect pests could be allowed to destroy the non-GM corn and would not develop resistance to B.t. pesticides. Gene transfer to weeds and other crops would not occur because the wind-blown pollen would not travel beyond the buffer zone. Estimates of the necessary width of buffer zones range from 6 meters to 30 meters or more. This planting method may not be feasible if too much acreage is required for the buffer zones.

Human health risks

 Allergenicity: Many children in the US and Europe have developed life-threatening allergies to peanuts and other foods. There is a possibility that introducing a gene into a plant may create a new allergen or cause an allergic reaction in susceptible individuals. A proposal to incorporate a gene from Brazil nuts into soybeans was abandoned because of the fear of causing unexpected allergic reactions. Extensive testing of GM foods may be required to avoid the possibility of harm to consumers with food allergies. Labeling of GM foods and food products will acquire new importance.

 Unknown effects on human health: There is a growing concern that introducing foreign genes into food plants may have an unexpected and negative impact on human health. A recent article published in Lancet examined the effects of GM potatoes on the digestive tract in rats. This study claimed that there were appreciable differences in the intestines of rats fed GM potatoes and rats fed unmodified potatoes. Yet critics say that this paper, like the monarch butterfly data, is flawed and does not hold up to scientific scrutiny. Moreover, the gene introduced into the potatoes was a snowdrop flower lectin, a substance known to be toxic to mammals. The scientists who created this variety of potato chose to use the lectin gene simply to test the methodology, and these potatoes were never intended for human or animal consumption. On the whole, with the exception of possible allergenicity, scientists believe that GM foods do not present a risk to human health.

Economic concerns

 Bringing a GM food to market is a lengthy and costly process, and of course agri-biotech companies wish to ensure a profitable return on their investment. Many new plant genetic engineering technologies and GM plants have been patented, and patent infringement is a big concern of agribusiness. Yet consumer advocates are worried that patenting these new plant varieties will raise the price of seeds so high that small farmers and third world countries will not be able to afford seeds for GM crops, thus widening the gap between the wealthy and the poor. It is hoped that in a humanitarian gesture, more companies and non-profits will follow the lead of the Rockefeller Foundation and offer their products at reduced cost to impoverished nations.
 Patent enforcement may also be difficult, as the contention of the farmers that they involuntarily grew Monsanto-engineered strains when their crops were cross-pollinated shows. One way to combat possible patent infringement is to introduce a "suicide gene" into GM plants. These plants would be viable for only one growing season and would produce sterile seeds that do not germinate. Farmers would need to buy a fresh supply of seeds each year. However, this would be financially disastrous for farmers in third world countries who cannot afford to buy seed each year and traditionally set aside a portion of their harvest to plant in the next growing season. In an open letter to the public, Monsanto has pledged to abandon all research using this suicide gene technology.