RAJALAKSHMI ENGINEERING COLLEGE Thandalam, Chennai 602 105 DEPARTMENT OF BIOTECHNOLOGY NOTES ON LESSON Faculty Name Subject Name

e Semester Class : Dr.B.Vijaya Geetha(Senior Lectuer) BT114 : PLANT BIOTECHNOLOGY : VII : IV SEC A& B

BT2030

PLANT BIOTECHNOLOGY Regulations 2008

LTPC 3003

AIM To develop the skills of the students in the area of Plant Biotechnology. OBJECTIVES At the end of the course the student would have learnt about the applications of Genetic Engineering in Plant and how to develop Transgenic plants. This will facilitate the student to take up project work in this area. Unit-I: Introduction to plant molecular biology Genomes are more than the sum of an organism's genes and have traits that may be measured and studied without reference to the details of any particular genes and their products. Researchers compare traits such as chromosome number (karyotype), genome size, gene order, codon usage bias, and GC-content to determine what mechanisms could have produced the great variety of genomes that exist today .Duplications play a major role in shaping the genome. Duplications may range from extension of short tandem repeats, to duplication of a cluster of genes, and all the way to duplications of entire chromosomes or even entire genomes. Such duplications are probably fundamental to the creation of genetic novelty. Horizontal gene transfer is invoked to explain how there is often extreme similarity between small portions of the genomes of two organisms that are otherwise very distantly related. Horizontal gene transfer seems to be common among many microbes. Also, eukaryotic cells seem to have experienced a transfer of some genetic material from their chloroplast and mitochondrial genomes to their nuclear chromosomes.

Genetic material of plant cell: 1.Nuclear genome. 2.Mitochondrial genome. 3.Chloroplast genome. 1.Nuclear genome Chromosomes and chromatin. Histones and chromatin. 5 major classes of histone proteins. Nucleosomes and nucleosome core particle. Structurial hierarchy of chromosomes.

Gene loci are non-randomly organized. This applies to their linear arrangements on chromosomes, as well as to their spatial organization in the nucleus. Recent chromosomewide and genome-wide studies give insights into which loci interact at the nuclear periphery with the lamina or nuclear pores. The functional role of peripheral localization in gene silencing is still unclear. Recent studies suggest that it regulates the silencing of some but not all loci. Active loci are enriched in the nuclear interior, and here they frequently associate with splicing speckles. Juxtaposition of chromosomal loci at such nuclear domains can falsely imply functional interactions. True functional interactions between chromosomal loci do, however, appear to regulate gene activity in many ways. 2. Mitochondrial genome Circular,simple, double stranded similar to prokaryotes. Contains genes codes for Oxidative phosphorylation, rRNA, tRNA.

Mitochondrial Genome Organization In comparison to the chloroplast genome, the size of the mitochondrial genome is quite variable. Species Oenothera Turnip Corn Muskmelon Size (kb) 195 218 570 2400

Further, in comparison to the mitochondrial genomes of other species the size is quite large and variable. For example, animal mitochondrial genomes range in size form 15-18 kb, and fungi mitochondrial genomes range form 18-78 kb. Plants may code for more proteins than with species. For example, genes for ribosomes, subunits I and II of cytochrome oxidase and ATPase subunits are located on the mitochondrial genomes of plants. When DNA from corn mitochondria was investigated with EM, several circular molecules of different sizes were detected. Once the genome was mapped it became apparent that a mechanism existed to generated these circles of different sizes. It is now understood how these molecules arise. First, lets look at the simple situation of turnip. Two direct repeats undergo intramolecular recombination to give the two smaller molecules: 218 kb -------> 135 kb + 83 kb The mitochondrial genome of corn undergoes the same type of recombination, but the events are more complex. First, the master circles can be subdivided into two major subgroups: 570 kb -------> 488 kb + 82 kb 570 kb -------> 503 kb + 67 kb The second group of molecules are still labile and can produce several other subpopulations. Further two subgenomic circles can unite to form a larger circle. This variability is possible because corn has 10 repeats with which intramolecular recombination can occur. Species Turnip Cauliflower Black Mustard White Mustard Radish Spinach Master Circle Size (kb) 218 217 231 208 242 327 Sub-genomic Circle Size (kb) 135 + 83 172 + 45 135 + 96 none 139 + 103 234 + 93 Repeat Size (kb) 2 ? 7 none 10 6

Introns have been located in the cytochrome oxidase subunit II gene (the cytochrome complex consists of 3 mitochondrial and 4 nuclear encoded genes). This gene contains one intron in rye, corn, wheat, rice, and carrot, but for other species such as Oenothera, broad bean, cucumber the gene has no intron. Promiscuous DNA

Stern and Lonsdale (1982) hybridized mtRNA to a SstII digest of maize mt DNA and found that it hybridized to fragments known not to contain mt rRNA genes. The question of interest was - what was it hybridizing to? They next looked at a cosmid clone of corn mtDNA that hybridized to the mt RNA and found that it hybridized to a RNA molecule of the size of the cp 16S RNA gene. How could this have happened? They next mapped the clone and compared it to the map of the corn cpDNA and found that the clone map was almost congruent with that of the the cpDNA 16S RNA region. This mapping showed that the two maps were nearly identical over a 12 kb region of DNA. These results suggest that cpDNA had been transferred to the mitochondrial genome. The observation that organelle DNA was found in other DNA compartments of the cell was extended by other researcher. Stern and Palmer looked at corn, mung bean, spinach and pea and found extensive evidence of cpDNA/mtDNA homology. These observations were extended to other DNA locations in the plant cellIt was demonstrated that mitochondrial DNA sequences are located in the nucleus of corn. Experiments showed that cpDNA sequences are found in the nuclear DNA. 3.Chloroplast genome Circular, simple, double stranded similar to prokaryotes. Contains genes codes for Rubisco enzymes,Cytochromes, rRNA, tRNA,Photosystem-I and photosystem II, and ATP synthase complex.

Chloroplast Genome Organization All angiosperms and land plants have cpDNAs which range in size from 120-160 kb; three expceptions are: Species N. accuminati Duckweed Geranium Size (kb) 171 180 217

All cpDNA molecules are circular and spinach is used as the basis for all comparisons. Very few repeat elements are found other than short sequences of less than 100 bp. The notable exception is a large (10-76 kb) inverted repeat section, which when present, always contains the rRNA genes. (Legumes such as pea do not contain this repeat.) For the majority of

species, this repeat region is 22-26 kb in size. Finally,the genetic order of the ribosomal unit is conserved in all species: 16S - tRNAile - tRNAala - 23S - 5S Recent research has also described two other features of chloroplast DNA. First it was shown to that it can exist in in two orientations This implies that the molecule can undergo an isomerization event. Second is has been shown that spinach, corn, tomato and pea can all exist as multimers . Multimer Monomer Dimer Trimer Tetramer Relative Abundance 1 1/3 1/9 1/27 Percent 67.5 22.5 7.5 2.5

Because photosysnthesis is the primary function of the chloroplast it is not surprising that the chlroplast genome contains genes which encode for proteins that are involved in that process. Reaction Function Dark Reactions rbcS (nuclear encoded) rbcL (chloroplast encoded) Light Reactions apoproteins for PSI andPSII cytochrome b6 cytochrome f 6 of 9 ATPase subunits cab, LHC proteins (nuclear encoded) plastocyanin (nuclear encoded) ferredoxin (nuclear encoded) Other 19/60 ribosome binding proteins translation factors RNA polymerase subunits tRNA and rRNA genes Atrazine resistance is apparantley mediated through the psbA gene sequences of the 32 kd protein which is encoded by cpDNA. DNA sequence analysis revealed the following amino acid changes that are thought to be important. Species AA# Susceptible Resistant Blue green algae 264 Ser (TCG) Ala (GCG)

Chlamydomonas 264 Ser (TCT) Ala (GCT) Solanum nigrum 264 Ser (AGT) Gly (GGT) Amaranthus 228 Ser (AGT) Gly (GGT) Evolutionary Changes of cpDNA 1. The majority of changes are small insertions and deletions of 1-106bp; significantly, a few length mutations of 50-1200 bp are clusted in "hot spots". 2. The largest deletion occured in pea where an entire rRNA cluster is lost. 3. The most common evolutionary change is in gene order. Small changes in the gene order occur, especially in the algae, but inversions have generated large scale order changes: o legumes - about 50 kb inversion brought rbcL closer to psbA
o

wheat - about 25 kb inversion brought atpA closer to rbcL

Junk and repeat sequences: Non coding sequences. Act as regulatory sequences. Types of Junk DNA Sequences Noncoding functional RNA Noncoding RNAs are functional RNA molecules that are not translated into protein. Examples of noncoding RNA include ribosomal RNA, transfer RNA, Piwi-interacting RNA and microRNA. MicroRNAs are predicted to control the translational activity of approximately 30% of all protein-coding genes in mammals and may be vital components in the progression or treatment of various diseases including cancer, cardiovascular disease, and the immune system response to infection. ''Cis''-regulatory elements Cis-regulatory elements are sequences that control the transcription of a gene. Cis-elements may be located in 5' or 3' untranslated regions or within introns. Promoters facilitate the transcription of a particular gene and are typically upstream of the coding region. Enhancer sequences may exert very distant effects on the transcription levels of genes.

Introns Introns are non-coding sections of a gene, transcribed to precursor mRNA, but ultimately removed by RNA splicing during the processing to mature messenger RNA. Many introns appear to be mobile genetic elements. Some introns do appear to have significant biological function, possibly through ribozyme functionality that may regulate tRNA and rRNA activity as well as protein-coding gene expression, evident in hosts that have become dependent on such introns over long periods of time; for example, the trnL-intron is found in all green plants and appears to have been vertically inherited for several billions of years, including more than a billion years within chloroplasts and an additional 23 billion prior in the cyanobacterial ancestors of chloroplasts. Pseudogenes that are the of retrotransposition of an RNA intermediate are known as processed pseudogenes; pseudogenes that arise from the genomic remains of duplicated genes or residues of inactivated are nonprocessed pseudogenes. and a substantial number of pseudogenes are actively transcribed. Repeat sequences, transposons and viral elements Transposons and retrotransposons are mobile genetic elements. Retrotransposon repeated sequences, which include long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), account for a large proportion of the genomic sequences in many species. Alu sequences, classified as a short interspersed nuclear element, are the most abundant mobile elements in the human genome. Some examples have been found of SINEs exerting transcriptional control of some protein-encoding genes. Endogenous retrovirus sequences are the product of reverse transcription of retrovirus genomes into the genomes of germ cells. Mutation within these retro-transcribed sequences can inactivate the viral genome. Approximately 8% of the human genome is made up of endogenous retrovirus sequences,, and as much as 25% is recognizably formed of retrotransposons. Genome size variation in at least two kinds of plants is mostly the result of retrotransposon sequences. Telomeres Telomeres are regions of repetitive DNA at the end of a chromosome, which provide protection from chromosomal deterioration during DNA replication. Functions of Junk DNA Many noncoding DNA sequences have very important biological functions. Comparative genomics reveals that some regions of noncoding DNA are highly conserved, sometimes on time-scales representing hundreds of millions of years, implying that these noncoding regions are under strong evolutionary pressure and positive selection.

For example, in the genomes of humans and mice, which diverged from a common ancestor 6575 million years ago, protein-coding DNA sequences account for only about 20% of conserved DNA, with the remaining majority of conserved DNA represented in noncoding regions. Some noncoding DNA sequences are genetic "switches" that do not encode proteins, but do regulate when and where genes are expressed. According to a comparative study of over 300 prokaryotic and over 30 eukaryotic genomes, eukaryotes appear to require a minimum amount of non-coding DNA. This minimum amount can be predicted using a growth model for regulatory genetic networks, implying that it is required for regulatory purposes. In humans the predicted minimum is about 5% of the total genome. Some specific sequences of noncoding DNA may be features essential to chromosome structure, centromere function and homolog recognition in meiosis. Some noncoding DNA sequences determine how much of a particular protein gets generated. Other sequences of noncoding DNA determine where transcription factors attach. Pseudogene sequences appear to accumulate mutations more rapidly than coding sequences due to a loss of selective pressure. What is Junk DNA In genetics, "junk DNA" or noncoding DNA describes components of an organism's DNA sequences that do not encode for protein sequences. In many eukaryotes, a large percentage of an organism's total genome size is noncoding DNA, although the amount of noncoding DNA, and the proportion of coding versus noncoding DNA varies greatly between species. Much of this DNA has no known biological function. However, many types of noncoding DNA sequences do have known biological functions, including the transcriptional and translational regulation of protein-coding sequences. Other noncoding sequences have likely but as-yet undetermined function, an inference from high levels of homology and conservation seen in sequences that do not encode proteins but appear to be under heavy selective pressure. Junk DNA Term Junk DNA, a term that was introduced in 1972 by Susumu Ohno, is a provisional label for the portions of a genome sequence of a for which no discernible function has been identified. According to a 1980 review in ''Nature'' by Leslie Orgel and Francis Crick, junk DNA has "little specificity and conveys little or no selective advantage to the organism".

The term is currently, however, a somewhat outdated concept, being used mainly in popular science and in a colloquial way in scientific publications, and may have slowed research into the biological functions of noncoding DNA. Several lines of evidence indicate that many "junk DNA" sequences have likely but unidentified functional activity, and other sequences may have had functions in the past. Still, a large amount of sequence in these genomes falls under no existing classification other than "junk". For example, one experiment removed 1% of the mouse genome with no detectable effect on the phenotype. This result suggests that the removed DNA was largely nonfunctional. In addition, these sequences are enriched for the heterochromatic histone modification H3K9me3.

Repetitive DNA sequences: Classified into 2 types: 1.Tandemly repetitive DNA. 2.Interspersed Repetitive DNA. Orgin of Repetitive DNA sequences. Mechanism of DNA amplification. 1.Unequal crossing over. 2.Rolling circle model. Function Repetitive DNA sequences: Act as regulatory sequences Subrepet sequences- Contain Promoter and Enhancers. Example : Major rRNA genes 18s ,5.8s ,25s rRNA ,5srRNA genes- Tandemly repetitive DNA.

Repeated Sequences The intermediate and fast components are composed of sequences that are found many times in the genome. These sequences are called repetitive sequences and can vary in size from a 100 bp to 1000 bp or more. Furthermore, these sequences have undergone sequence divergence by the addition or deletion of sequences or by changes in the base pair sequence. Thus, the repeated sequences themselves show some divergence. An example of a highly repetitive sequence is the repeat found to be associated with the knob heterochromatin of corn. It ranges from 3-5 x 105 copies on a small knob to 1 x 10^6 on the large knobs. This sequence is unique to knobs and is not found associated with any other heterochromatic regions of corn. An example of a functional repeated sequence in plants is the corn storage proteins, zeins. Two major classes of zeins exist, the 22 and 19 kd classes. Sequence analysis has shown that both classes have the same structure. Signal Sequence ----- Head ----- Repeat Unit -----Tail The only difference between the 22 and 19 kd class is the repeat unit. The 22 kd class has 8 repeat units and the 19 kd class has 7 repeat units. Estimates have been made of the number of copies of these genes and 30-50 copies of the 22 kd class are found in the corn genome. Thus, the repeat unit would be represented 240-400 times in the genome. Transcription in eukaryotes: Mediated by 3 RNA Polymerase namely 1.RNA polymerase-I : used for synthesis of most rRNAs. 2 RNA polymerase-II : used for synthesis of mRNAs. 3. RNA polymerase-III : used for synthesis of 5sRNAs,tRNA ,nuclear and cytosolic rRNAs. Mechanism of Transcription: Includes 3 steps 1.Initiation.-Promoter sequence- TATA box,CAATbox. 2.Elongation-Transcription bunnle. 3.Termination.

Translation in eukaryotes: Includes 3 steps 1.Initiation -8IF are required..mRNA,initiator tRNA,80s ribosomes.

2.Elongation -3 elongation factors are required. 3.Termination single release factor.

Unit II Chloroplast and Mitochondria. Structure of chloroplast: Consist of outermembrane, Innermembrane. Thylakoids, Stroma, Thylakoid membrane.

Chloroplast are those sub-units in a plant cell, which produce food for a plant through the process of photosynthesis. Chloroplasts are somewhat similar to mitochondria, a single celled organisms which can produce energy. Chloroplasts reproduce by the process of division of cells and have their own genetic systems. It's the chloroplasts which convert carbon di-oxide into carbohydrates, which are consumed by plants. Moreover amino acids, lipid components and fatty acids of the cell membranes are synthesized by chloroplasts. In addition to that, chloroplasts reduce nitrogen into ammonia and other organic compounds. Once you have got a basic idea of chloroplast function, let's take a look at how they function in a plant cell. Chloroplast Structure The chloroplasts are usually 5-10 micrometer long and consist of circular DNA molecules. They are bound by a two-layered membrane which is known as the chloroplast envelope. Besides these membranes, they have another membrane which is known as the thylakoid

membrane. This membrane has numerous flat disc like structures, called the thylakoids and they are arranged in piles called the grana. The internal composition of the chloroplast is highly complex due its membrane structure. The chloroplast is divided into three compartments - the thylakoid lumen, which is the outer-most membrane, the stroma the middle membrane and the intermembrane space, the space between a chloroplasts' two membranes. Even though the structure is highly complex, the functions of each of these membranes are not that complex. The outermost membrane of the chloroplast envelope contains porins which absorb sunlight. They also contain small molecules which can easily penetrate the membranes and transport sun's energy. On the other hand, the inner membrane or the stroma is not permeable to metabolites and ions, thereby trapping sun's energy in them. The stroma contains a number of metabolic enzymes, which are responsible for converting carbon dioxide to carbohydrates during the process of photosynthesis. If seen under an electronic microscope, the thylakoid membranes look like alternate lightand-dark bands and each of them are 0.01 micrometer thick. These membranes consist of some antenna complexes and it's these which contain chlorophyll besides protein, which combines these pigments together. The antenna complexes increase the area of the chloroplasts, thereby allowing them to capture more light, besides capturing photons with higher wavelengths. Energy absorbed by these photons is then captured and transferred to the center of the chloroplast. The chlorophyll molecules gets ionized, thereby producing excited electrons, which in turn passes light energy to the stroma, so as to produce food. Chloroplast Function in a Plant Cell Chloroplast contain an important component of a plant cell called chlorophyll, which is responsible for production of food. It's the chlorophyll which gives the leaves its characteristic green color. It's in the chloroplasts where chemical reactions take place for the production of foods. Chlorophyll contained in the chloroplasts is responsible for absorbing sunlight. It's through the process of photosynthesis that a plant makes food for itself. The process includes absorbing the energy from the sun so as to create sugar. When sunlight hits a chloroplast, the chlorophyll in it uses the energy and in combination with carbon dioxide and water forms sugar and oxygen. Plants use these sugars for survival and the oxygen released is used by animals to breathe.

Structure of Mitochondria:
Consist of Outermembrane, Innermembrane.-site for ATP generation. Intermembrane space

Matrix -consist of enzymes for TCA cycle Cristae

Structure of Mitochondria The cytoplasm of nearly all eukaryotic cells contain mitochondria, although there is at least one exception, the protist Chaos (Pelomyxa) carolinensis. They are especially abundant in cells and parts of cells that are associated with active processes. For example, in flagellated protozoa or in mammalian sperm, mitochondria are concentrated around the base of the flagellum or flagella. In cardiac muscle, mitochondria surround the contractile elements. Hummingbird flight muscle is one of the richest sources of mitochondria known. Thus, from their distribution alone one would suspect that they are involved in energy production. Multicellular organisms probably could not exist without mitochondria. The inability to remove electrons from the system and the buildup of metabolic end products restrict the utility of anaerobic metabolism. Through oxidative phosphoryation mitochondria make efficient use of nutrient molecules. They are the reason that we need oxygen at all. The double-membraned mitochondrion can be loosely described as a large wrinkled bag packed inside of a smaller, unwrinkled bag. The two membranes create distinct compartments within the organelle, and are themselves very different in structure and in function.

The outer membrane is a relatively simple phospholipid bilayer, containing protein structures called porins which render it permeable to molecules of about 10 kilodaltons or less (the size of the smallest proteins). Ions, nutrient molecules, ATP, ADP, etc. can pass through the outer membrane with ease. The inner membrane is freely permeable only to oxygen, carbon dioxide, and water. Its structure is highly complex, including all of the complexes of the electron transport system, the ATP synthetase complex, and transport proteins. The wrinkles, or folds, are organized into lamillae (layers), called the cristae (singlular: crista). The cristae greatly increase the total surface area of the inner membrane. The larger surface area makes room for many more of the above-named structures than if the inner membrane were shaped like the outer membrane.

The membranes create two compartments. The intermembrane space, as implied, is the region between the inner and outer membranes. It has an important role in the primary function of mitochondria, which is oxidative phosphorylation. The matrix contains the enzymes that are responsible for the citric acid cycle reactions. The matrix also contains dissolved oxygen, water, carbon dioxide, the recyclable intermediates that serve as energy shuttles, and much more (see "other functions"). Diffusion is a very slow process. Because of the folds of the cristae, no part of the matrix is far from the inner membrane. Therefore matrix components can diffuse to inner membrane complexes and transport proteins within a relatively short time. Electron micrographs have revealed the three dimensional structure of mitochondria. However, since micrographs are themselves two dimensional, their interpretation can be misleading. Texts frequently show a picture of a 'typical' mitochondrion as a bacteria-sized ellipsoid (perhaps 0.5 by 1 micrometer). However, they vary widely in shape and size. Electron micrographs seldom show such variation, because they are two-dimensional images.

Isolated mitochondria, such as from homogenized muscle tissue, show a rounded appearance in electron micrographs, implying that mitochondria are spherical organelles.

Mitochondria in situ can be free in the cytoplasm or packed in among more rigid structures, such as among the myofibrils of cardiac muscle tissue. In cells such as muscle, it is clear that mitochondria are not spherical, and often are not even ellipsoid. In some tissues, the mitochondria are almost filamentous, a characteristic that two dimensional micrographs may fail to reveal.

Function of Mitochodria: 1.Electron Transport Chain 2. Generation of ATP by Oxidative Phosphorylation. Additional functions as:

Regulation of the membrane potential Apoptosis-programmed cell death Calcium signaling (including calcium-evoked apoptosis Cellular proliferation regulation Regulation of cellular metabolism Steroid synthesis.

Rubisco synthesis: Consist of 8 large 55kd (L)and 8 small 13kd subunits(S). Each L chain contains catalytic site and regulatory site- synthesised by chloroplast genes. Each S chain contains- Regulatory subunits-direct the synthesis of rubisco by binding to a regulatory siteof L chain.-synthesised by nuclear genes. RUBISCO

Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) catalyzes the first step in net photosynthetic CO2 assimilation and photorespiratory carbon oxidation. The enzyme is notoriously inefficient as a catalyst for the carboxylation of RuBP and is subject to competitive inhibition by O2, inactivation by loss of carbamylation, and dead-end inhibition by RuBP. These inadequacies make Rubisco rate limiting for photosynthesis and an obvious target for increasing agricultural productivity. Resolution of X-ray crystal structures and detailed analysis of divergent, mutant, and hybrid enzymes have increased our insight into the structure/function relationships of Rubisco. The interactions and associations relatively far from the Rubisco active site, including regulatory interactions with Rubisco activase, may present new approaches and strategies for understanding and ultimately improving this complex enzyme.

INTRODUCTION As the entry point of CO2 into the biosphere, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is central to life on earth. Its very slow catalytic rate of a few per second, the low affinity for atmospheric CO2, and the use of O2 as an alternative substrate for the competing process of photorespiration together make Rubisco notoriously inefficient as the initial CO2-fixing enzyme of photosynthesis. Consequently, land plants must allocate as much as 50% of their leaf nitrogen to Rubisco, making this single enzyme the most abundant protein in the world. As the rate-limiting step of photosynthesis in both C3 and C4 plants, Rubisco is often viewed as a potential target for genetic manipulation to improve plant yield. The food, fiber, and fuel needs of an ever-increasing human population and shortages in the availability of water for agriculture are challenges of the twenty-first century that would be impacted positively by successful manipulation of Rubisco in crop plants. During the past 10 years, resolution of a variety of Rubisco atomic structures has increased our understanding of the reaction mechanism of the enzyme. Based on this information, the new conventional wisdom is that improving Rubisco will not be simple but will require multiple mutations that subtly change the positioning of critical residues within the active site. Considering the explosion of new knowledge that has occurred in just the past few years regarding the interactions and associations that impact the structure, function, and regulation of Rubisco, there is reason to believe that successful manipulation of Rubisco may yet be achieved. GENERAL STRUCTURAL AND FUNCTIONAL CONSIDERATIONS Wealth of Rubisco Structures and Sequences Besides being one of the slowest, Rubisco is also one of the largest enzymes in nature, with a molecular mass of 560 kDa. In land plants and green algae, the chloroplast rbcL gene encodes the 55-kDa large subunit, whereas a family of rbcS nuclear genes encodes nearly identical 15-kD small subunits. Following posttranslational processing of both subunits,

small subunits are added to a core of chaperone-assembled large subunits in the chloroplast. The resulting Form I Rubisco holoenzyme is composed of eight large and eight small subunits. Variations on this theme include the Form II Rubisco of some prokaryotes and dinoflagellates consisting of a dimer of only large subunits and the Form I Rubisco of nongreen algae produced from rbcL and rbcS genes that are both chloroplast encoded. In still another variation, the Rubisco from archaebacteria is neither Form I or II, but a decamer comprising five large-subunit dimers. More than 20 Rubisco X-ray crystal structures now exist within the Protein Data Bank.. These range from the homodimeric holoenzyme ofRhodospirillum rubrum that provided the first glimpse of the active site to the high-resolution structures of spinach Rubisco with bound substrate, product, and transition-state analogs. The C-terminal domain of the large subunit of every Rubisco enzyme forms a classic /-barrel. Residues predominately in the loops between strands and helices interact with the transition-state analog 2-carboxyarabinitol 1,5-bisphosphate (CABP). Because several N-terminal-domain residues of a neighboring large subunit also participate as designated active-site residues, the functional unit structure of Rubisco is a large-subunit dimer. Four pairs of associated large subunits are capped on each end by four small subunits, each of which interacts with three large subunits. Because large subunits of Form II enzymes contain all the structural elements required for catalysis, the origin and role of the small subunit in Form I enzymes remain enigmatic. More than 2000 rbcL and 300 rbcS sequences now reside within GenBank, generated primarily for phylogenetic reconstruction. The deduced large-subunit sequences are fairly conserved, and any differences in length occur primarily at the N and C termini. (Throughout this review, numbering of large-subunit residues will be based on the sequence of the spinach large subunit.) rbcL-like sequences have also been found in prokaryotes that do not possess photoautotrophy via the Calvin cycle. However, the deduced products of these genes appear to lack certain residues essential for carboxylation, indicating that they may have other functions in the cell. Small subunits are more divergent than large subunits. (Throughout this review, numbering of small-subunit residues will be based on species-specific sequences.) Whereas land-plant and green-algal small subunits generally have larger loops between strands A and B, some prokaryotes and all nongreen algae have longer C-terminal extensions. Small but significant regions of sequence identity have been observed between theSynechococcus small subunit and a protein associated with carboxysome assembly, indicating that there may be a shared interaction with the Rubisco large subunit or divergence from a common ancestral protein of unknown function. Interactions in larger subunit Whereas all Rubisco X-ray crystal structures show very similar C backbone structures, there is substantial divergence in amino-acid side chains. Thus, it is difficult to correlate

differences in structure with differences in kinetic properties. It has also been a challenge to apply genetic methods for investigating divergent Rubisco enzymes. Because eukaryotic Rubisco holoenzymes fail to assemble when their subunits are expressed inEscherichia coli , most directed mutations have been made in the R. rubrum or Synechococcus Rubisco enzymes expressed inE. coli . Only in tobacco and the green algaChlamydomonas reinhardtii has it become possible to transform the chloroplast genome as a means for analyzing the effects of mutant large subunits on the function of eukaryotic Rubisco. Although progress has been made in eliminating or substituting rbcL genes in photosynthetic prokaryotes, these systems have yet to be exploited for the genetic dissection of Rubisco structure/function relationships.

Mutational Approaches The first attempt to step outside the sphere of the active-site residues was made using classical genetics with Chlamydomonas . Because this organism can survive in the absence of photosynthesis when supplied with acetate as a source of carbon and energy and continues to synthesize a complete photosynthetic apparatus even when grown in darkness, a number of rbcL missense mutants were recovered by screening acetate-requiring strains (G54D, G171D, T173I, R217S, G237S, L290F, V331A). Four of the missense mutants (G54D, R217S, L290F, V331A) and their suppressors have defined regions relatively far from the active site that can influence . These regions include the secondary structural elements close to the loops that contain Lys-201 and Lys-334, as well as regions buried within the Nterminal domain and at the interface between large and small subunits. Despite the fact that Rubisco is required for the survival of land plants, two rbcL missense mutations (S112F and G322S) that disrupt holoenzyme assembly have been identified in variegated mutants of tobacco. Missense mutations recovered by screening are not distributed randomly within the gene because only those that affect essential structural or functional properties will be observed. Thus, most of the resulting amino-acid substitutions are likely to provide useful information about catalysis or assembly. For reasons that are not readily apparent, no rbcL structural-gene mutation has yet been described from the screening of photosynthesis-deficient prokaryotes. Scanning mutagenesis, in which all residues of a certain type are replaced by directed mutagenesis, has been used to examine those Gly residues that are conserved among all Rubisco large subunits . When each of the 22 Gly residues in the Synechococcus enzyme was replaced with either Ala or Pro, only the G47A, G47P, G122A, G171A, G179A, G403A, G405A, and G416A enzymes retained some carboxylase activity. However, in the absence of detailed kinetic analysis, it is not known whether further study of the regions affected by these substitutions would be informative. Other conserved large-subunit residues distant from

the active site have been replaced by directed mutagenesis with effects on Rubisco function or stability. For example, when Cys-172 was replaced with Ser in the Chlamydomonas large subunit, an increase in holoenzyme stability in vivo was observed under oxidative stress conditions that triggered holoenzyme degradation. However, as noted above, replacement of conserved residues is not expected to account for differences in catalysis among divergent enzymes. Most directed mutagenesis studies aimed at examining distant interactions have relied on a phylogenetic approach in which the identities of residues are changed from those of one species to those of another species of Rubisco. The few conserved differences in sequences between C3 and C4 plant Rubisco and between Rubisco enzymes that display different specificities for interaction with Rubisco activase have been identified. However, because a variety of land-plant Rubisco enzymes cannot be genetically engineered, conclusions must be based primarily on alterations of the Synechococcus or Chlamydomonas enzyme . The major limitation to the phylogenetic approach is, once again, deciding which of the many divergent regions are worth analyzing. Nonetheless, by combining this approach with others, two largesubunit regions have been investigated intensely. One of these regions is close to the active site, and the other is distant. Interactions in smaller subunit The existence of Form II Rubisco enzymes, composed of only large subunits, indicates that small subunits are not absolutely essential for carboxylase activity. Furthermore, Synechococcus large-subunit octamers (void of small subunits) retain 1% carboxylase activity and have a normal value. However, Vc is drastically reduced in such minimal enzymes, and a variety of side products are produced by misprotonation of RuBP. Scanning mutagenesis of cyanobacterial small subunits, expressed with large subunits in E. coli, has also shown that substitutions at some of the conserved residues can decrease Vc and holoenzyme assembly. Although it is apparent that small subunits can influence catalysis indirectly, it has been difficult to determine whether divergent small-subunit residues play a role in the differences in kinetic constants among Rubisco enzymes from different species. R. rubrum Rubisco lacks small subunits, small subunits of nongreen algae are encoded by the chloroplast genome (which has yet to be transformed), and land plants have a family of rbcS genes in the nucleus that cannot be readily eliminated . ACTIVASE ISOFORMS An interesting but complicating feature of activase is the presence of two subunits of approximately 47 kDa and 42 kDa in many plant species. In all cases that have been reported to date, alternative splicing of a pre-mRNA produces these two proteins that are identical except for the presence of an extra 2736 amino acids at the C-terminus of the longer form

.The two activase polypeptides are active both in ATP hydrolysis and Rubisco activation, but they differ in kinetic properties, as well as in their thermal stabilities. Activase as a Potential Target for Increasing Photosynthesis Because most strategies for improving Rubisco require a change in the structure of the enzyme, it is necessary to consider how each change will affect the interaction of Rubisco with activase, chaperonins, and other proteins necessary for assembly or function. For example, improvements that involve replacement of Rubisco subunits, even if properly assembled, could be ineffective in vivo if activase is unable to recognize the Rubisco and reverse formation of dead-end complexes This problem might even occur with single aminoacid substitutions if they alter recognition of Rubisco by activase. Thus, each strategy for improving Rubisco should be mindful of the possible need to co-design activase. Redesigning activase will require a more complete understanding of the mechanism of action and the sites for interaction with Rubisco. Because the activation state of Rubisco limits photosynthesis under conditions of high CO2 and temperature, improvements in activase may stimulate photosynthesis under certain conditions. For example, the decrease in Rubisco activation that occurs in response to elevated CO2appears to involve limitation of activase by [ATP]. Engineering activase to be less sensitive to inhibition by ADP, either by directed mutagenesis or by altering the relative expression of the two forms, may improve the performance of plants under high CO2. At elevated temperatures, Rubisco activation decreases to levels that limit photosynthesis because activase is unable to keep pace with the much faster rate of Rubisco deactivation. The poor performance of activase at high temperature is caused, in part, by its exceptional thermal lability. Thus, changes in activase that improve its thermal stability or increase its amount represent possible approaches for increasing photosynthesis at elevated temperatures. The latter approach may be especially useful in C4 plants, which have elevated levels of CO2 at the site of Rubisco but do not exhibit a marked stimulation of photosynthesis by temperature because of lower Rubisco activation (S. J. Crafts-Brandner & M. E. Salvucci, unpublished). PROSPECTS FOR IMPROVEMENT Gross changes in the gaseous composition of the earth's atmosphere has selected for landplant Rubisco with a relatively high . By comparison, the evolutionarily pressure to optimize the land-plant enzyme for performance in controlled agricultural settings has been minimal and indirect. Instead, land plants have evolved under natural conditions where the availability of water and/or nitrogen often limits photosynthesis. As shown by the many studies involving CO2-enrichment, increasing the rate of carboxylation by Rubisco will increase plant yield, provided that sufficient nitrogen is available for increased protein synthesis. Improving the catalytic efficiency of Rubisco will have the same effect but require less nitrogen to implement.

As the new century opens, the prospects for improving Rubisco are excellent. Elucidation of the X-ray crystal structures of Rubisco in its many forms has provided a structural framework for understanding Rubisco function and evaluating the effects of mutations. Coupled with thousands of Rubisco sequences, these structures may guide phylogenetic and bioinformatic inquiries into the diversity of kinetic parameters. Successful efforts to produce mutations in the chloroplast-encoded large subunit and to replace subunits have laid the groundwork for applying new approaches to understanding Rubisco structure/function relationships. Finally, the realization that Rubisco function is tied to and can be limited by its interaction with activase indicates that improvements in activase may provide a totally new approach for enhancing photosynthesis.

Regulation of Rubisco: Rubisco is regulated by 1.Regulation by ions.-Mg 2+ ions. 2.Regulation by Activase. 3.Regulation by Phosphate. 4.Regulation by carbondioxide. Transport of Mitochondrial proteins: Proteins Oxidative phosphorylation ,Enzymes required for TCA cycle ,Replication and expression are nuclear encoded. Proteins targeted to mitochondria 20 to 35 + charged aminoacids at the aminoterminal endPresequences. Mitochondrial protein import requires 1.ATP 2.Electric potencial across the membrane. Proteins are targeted to 1.mitochondrial inner membrane. 2.mitochondrial matrix 3.outermembrane. 4.Intermembrane space.

Cytoplasmic male sterility: Occurs due to mutation in the mitochondrial genome-which causes sterility in the plants. Cytoplasmic male sterility is the total or partial male sterility associated with plant biology as the result of specific nuclear and mitochondrial interactions. Male sterility is the failure of plants to produce functional anthers, pollen, or male gametes. The first documentation of male sterility came in Joseph Gottlieb Klreuter observed anther abortion within species and specific hybrids. Cytoplasmic male sterility has now been identified in over 150 plant species. It is more prevalent than female sterility, either because the male sporophyte and gametophyte are less protected from the environment than the ovule and embryo sac, or because it results from natural selection on mitochondrial genes which are maternally inherited and are thus not concerned with pollen production. Male sterility is easy to detect because a large number of pollen grains are produced and are easily studied. Male sterility is assayed through staining techniques (carmine, lactophenol or iodine); while detection of female sterility is detectable by the absence of seeds. Male sterility has propagation potential in nature since it can still set seed and is important for crop breeding, while female sterility does not. Male sterility can be aroused spontaneously via mutations in nuclear and/or cytoplasmic genes. Male sterility can be either cytoplasmic or cytoplasmic-genetic. Cytoplasmic male sterility (CMS) is caused by the extranuclear genome (mitochondria or chloroplast) and shows maternal inheritance. Manifestation of male sterility in CMS may be either entirely controlled by cytoplasmic factors or by the interaction between cytoplasmic and nuclear factors. Cytoplasmic male sterility Cytoplasmic male sterility, as the name indicates, is under extra-nuclear genetic control (under the control of the mitochondrial or plastid genomes). They show non-Mendelian inheritance and are under the regulation of cytoplasmic factors. In this type, male sterility is inherited maternally. In general there are two types of cytoplasm: N (normal) and the aberrant S (sterile) cytoplasms. These types exhibit reciprocal differences. Cytoplasmic-genetic male sterility While CMS is controlled by an extranuclear genome often times nuclear genes can have the capability to restore fertility. When nuclear restorations of fertility genes (Rf) are available

for CMS system in any crop, it is cytoplasmic-genetic male sterility; the sterility is manifested by the influence of both nuclear (Mendelian inheritance) and cytoplasmic (maternally inherited) genes. There are also restorers of fertility (Rf) genes, which are distinct from genetic male sterility genes. The Rf genes do not have any expression of their own unless the sterile cytoplasm is present. Rf genes are required to restore fertility in S cytoplasm which causes sterility. Thus N cytoplasm is always fertile and S cytoplasm with genotype Rfproduces fertiles; while S cytoplasm with rfrf produces only male steriles. Another feature of these systems is that Rf mutations (i.e., mutations to rf or no fertility restoration) are frequent, so N cytoplasm with Rfrf is best for stable fertility. Cytoplasmic-genetic male sterility systems are widely exploited in crop plants for hybrid breeding due to the convenience to control the sterility expression by manipulating the gene cytoplasm combinations in any selected genotype. Incorporation of these systems for male sterility evades the need for emasculation in cross-pollinated species, thus encouraging cross breeding producing only hybrid seeds under natural conditions. Cytoplasmic male sterility in hybrid breeding Hybrid production requires a female plant in which no viable male gametes are borne. Emasculation is done to make a plant devoid of pollen so that it is made female. Another simple way to establish a female line for hybrid seed production is to identify or create a line that is unable to produce viable pollen. This male sterile line is therefore unable to selfpollinate, and seed formation is dependent upon pollen from the male line. Cytoplasmic male sterility is used in hybrid seed production. In this case, the sterility is transmitted only through the female and all progeny will be sterile. This is not a problem for crops such as onions or carrots where the commodity harvested from the F1 generation is produced during vegetative growth. These CMS lines must be maintained by repeated crossing to a sister line (known as the maintainer line) that is genetically identical except that it possesses normal cytoplasm and is therefore male fertile. In cytoplasmic-genetic male sterility restoration of fertility is done using restorer lines carrying nuclear restorer genes in crops. The male sterile line is maintained by crossing with a maintainer line which has the same genome as that of the MS line but carrying normal fertile cytoplasm. Cytoplasmic male sterility in hybrid maize breeding Cytoplasmic male sterility is an important part of hybrid maize production. The first commercial cytoplasmic male sterile, discovered in Texas, is known as CMS-T. The use of CMS-T, starting in the 1950s, eliminated the need for detasseling. In the early 1970s plants containing CMS-T genetics were susceptible to southern corn leaf blight and suffered from

widespread loss of yield. Since then CMS types C and S are used instead. Unfortunately these types are prone to environmentally induced fertility restoration and must be carefully monitored in the field. Environmentally induced restoration is when certain environmental stimuli signal the plant to bypass sterility restrictions and produce pollen anyway. Environmentally induced restoration differs from genetic restoration in that it is rooted in external signals rather than genetic DNA. The systematic sequencing of new plant species in recent years has uncovered the existence of several novel RF genes and their encoded proteins. A unified nomenclature for the RF extended protein families across all plant species, fundamental in the context of comparative functional genomics. This unified nomenclature accommodates functional RF genes and pseudogenes, and offers the flexibility needed to incorporate additional RFs as they become available in future. Unit-III:Plant metabolism and metabolic Engineering: Nitrogenase enzyme: Responsible for nitrogen fixation. Consist of 2 subunits namely 1.Mo-Fe containg Proteins 2.Fe- Containing Proteins. Requirement for nitrogen fixation: 1.Nitrogenase and Hydrogenase enzyme. 2.ferrodoxin. 3.Electron donor-Pyruvic acid or sucrose or glucose. 4.ATP. 5.Coenzyme and cofactor,Inorganic Phosphate and Mg2+ ions. 6.A carbon compound trapping ammonia. Nitrogenase (EC 1.18.6.1) is the enzyme used by some organisms to fix atmospheric nitrogen gas (N2). It is the only known family of enzymes which accomplishes this process. Dinitrogen is quite inert because of the strength of its N-N triple bond. To break one nitrogen atom away from another requires breaking all three of these chemical bonds. Whilst the equilibrium formation of ammonia from molecular hydrogen and nitrogen has an overall negative enthalpy of reaction (H0 = -45.2 kJ mol1 NH3), the energy barrier to activation is generally insurmountable (EA = 420 kJ mol1) without the assistance of catalysis.

The enzyme therefore requires a great deal of chemical energy, released from the hydrolysis of ATP, and reducing agents, such as dithionite in vitro or ferredoxin in vivo. The enzyme is composed of the heterotetrameric MoFe protein that is transiently associated with the homodimeric Fe protein. Nitrogenase is supplied reducing power when it associates with the reduced, nucleotide-bound homodimeric Fe protein. The heterocomplex undergoes cycles of association and disassociation to transfer one electron, which is the limiting step in the process. ATP supplies the reducing power. Each electron transferred supplies enough energy to break one of dinitrogen's chemical bonds, though it has not yet been proven that exactly three cycles are sufficient to convert one molecule of N2 to ammonia. Ultimately, nitrogenase bonds each atom of nitrogen to three hydrogen atoms to form ammonia (NH3), which is in turn bonded to glutamate to form glutamine. The exact mechanism of catalysis is unknown due to the difficulty in obtaining crystals of nitrogen bound to nitrogenase. This is because the resting state of MoFe protein does not bind nitrogen and also requires at least three electron transfers to perform catalysis. Nitrogenase is able to reduce acetylene, but is inhibited by carbon monoxide, which acts competitively, blocking the active site from dinitrogen. Dinitrogen will prevent acetylene binding, but acetylene does not inhibit binding of dinitrogen and only requires only one electron for reduction.[2] All nitrogenases have an iron- and sulfur-containing cofactor that includes a heterometal complex in the active site (e.g. FeMoCo). In most, this heterometal has a central molybdenum atom, though in some species it is replaced by a vanadium atom or iron. Due to the oxidative properties of oxygen, most nitrogenases are irreversibly inhibited by dioxygen, which degradatively oxidizes the Fe-S cofactors. This requires mechanisms for nitrogen fixers to avoid oxygen in vivo. Despite this problem, many use oxygen as a terminal electron acceptor for respiration. One known exception is the nitrogenase of Streptomyces thermoautotrophicus, which is unaffected by the presence of oxygen. The Azotobacteraceae are unique in their ability to employ an oxygen-labile nitrogenase under aerobic conditions. This ability has been attributed to a high metabolic rate allowing oxygen reduction at the cell membrane, but this idea has been shown to be unfounded and impossible at oxygen concentrations above 70 M (ambient concentration is 230 M O2), as well as during additional nutrient limitations. The reaction that this enzyme performs is: N2 + 8 H+ + 8 e + 16 ATP 2 NH3 + H2 + 16 ADP + 16 Pi Mechanism of nitrogen fixation: Includs 2 steps

1.Electron activation- Fe protein subunit. 2.Subrate reduction-Mo-Fe protein. Nif genes: Occurs as cluster of chromosomal genes of nitrogen fixing bacteria. Nif genes encodes for Nitrogenase complex and other enzymes involved in nitrogen fixation. Nearly 21 cluster of nif genes are identified. Nif genes H,D,K codes for the enzyme nitrogenase . Nif genes structure, activity and regulation

Nif genes which encode the nitrogenase complex and other enzymes involved in nitrogen fixation has consensus sequences identical from one nitrogen fixing bacteria to another, but while the structure of the nif genes is similar, the regulation of the nif genes varies between different diazotrophes (=nitrogen fixing organisms), depending also upon the organism's evolutionary hierarch. The nitrogenase complex is encoded by approximately 20 different nif genes. Summarized in the table bellow are the nif genes products and their role in nitrogen fixation

Activation of the nif genes transcription takes place in times of nitrogen stress. In most plants, activation of nif genes transcription is done by the nitrogen sensitive NifA protein. When there isn't enough fixed nitrogen factor for available for the plant's use, NtrC which is a RNApolymerase triggers NifA's expression, and NifA activates the rest of the nif genes transcription. If there is a sufficient amount of reduced nitrogen or oxygen is present, another protein is activated NifL, and NifL inhibits NifA activity resulting in the inhibition of nitrogenase forming. NifL is regulated by glnD and glnK gene products. The nif genes can be found on bacteria's chromosomes, but a lot of the times they are found on bacteria's plasmids with other genes related to nitrogen fixation (such as the nod genes).

Nod genes: Present in symbiotic nitrogen fixing bacteria. Responsible for nodule formation in root hairs of plants. Nod genes include nod ABC- codes-.Root hair curling nod D codes interact with flavonoid compound of plants and induces trancription of nod ABC genes.

nod H,Q,E,F encodes host specic nodulation. Bacteroids: Root Nodule

Formation

in

Rhizobium-Legume

Association

Rhizobium occurs free-living in the soil but does not fix nitrogen in this situation. It has recently been studied to fix nitrogen in the laboratory, in the absence of host when supplied with carbolic acid, pentose and small amount of fixed nitrogen. This confirms that it is Rhizobium itself that contains all necessary genetic information for fixing nitrogen but does not imply so, when living-free in the normal soil environments; its association with leguminous roots and formation of nodules seems obligatory to fix nitrogen. The stages envolved in root nodule formation are now fairly understood and include (i) recognition and attachment, (ii) penetration and travel, (iii) bacteroid formation and development of mature nodule. (i) Recognition and Attachment

In response to the variety of organic metabolites secreted by the roots of legume plants, the rhizobia migrate towards and grow in the rhizosphere and build up to high population density. It is considered that a series of flavonoid signals are there in organic metabolites that lead to the exchange of recognition signals thus attracting specific rhizobia species to specific legume roothairs. However, all species of Rhizobium (and Bradyrhizobium) possess specific adhesion protein called rhicadhesin on their surface. Rhicadhesin is a calcium-binding protein and binds calcium complexes on the surface of root hairs. Lectins, carbohydrate containing proteins, also contribute in RhizobiumLegume attachment.

1. Migration and growth of rhizobia in 2. Attachment of Rhizobia on Root Hair and Root rhizosphere Hair Curling and Penetration by Rhizobium

3. Infection Thread Growth 4. Cell to Cell Spread of Rhizobium and Rhizobial Travel

5. Bacteroids Differentiation Bacteroids Symbiosomes

and

the of into

6. T.S. of a Single Root Nodule

7. Mature Nodules on Legume Root

(ii) Penetration and Travel After attachment, the root hair curls as a result of the action of substances, excreted by the Rhizobium species called nod-factors. Some physiologists believe that curling is also affected by indole acetic acid, a plant growth hormone. After curling of the root-hair, the bacteria penetrate and enter the root-hair and induce the plant to develop a cellulosic tube, called infection thread, which extends inward to the root-hair . The Rhizobium cells then spread within the infection thread, move into the underlying root cells, and are released into cytoplasm of the host cell through the action of an organizer produced by the interaction between the rhizobial polysaccharides and component of root cells. Nod factors now stimulate root cell division eventually leading to the development of the root nodule. (iii) BBacterial Formation and Development of Mature Nodule When the bacteria are released from the infection thread into the host cell cytoplasm, they get

transformed into swollen, irregular-shaped, branched structures called bacteroids which then become surrounded singly or in small groups by a plant-derived membrane, called peribacteroid membrane, to form structures called 'symbiosome'. Symbiosomes are, in fact, the sites of nitrogen fixation. At this point, however, the symbiosomes secrete a hormone which enables the polyploid cells to divide rapidly forming the core of the nodule and the surrounding diploid cells also to divide and differentiate to cover the nodule in cortical tissue and to form vascular connections with the root. This hormone also enables the production of leghaemoglobin which protects the nitrogen fixation enzymes from oxygen. Other specific nodule components are also produced to complete the nodulation process.

Flavonoids Induce Nodulation In the last decade, the following root secreted flavonoids from leguminous plants have been known to induce transcription of nodulation (nod) genes: Plant source Alfalfa seed (Medicago sativa) Clover seedling (Trifolum spp.) Soybean seeding (Glycine max) Bean seed (Phaseolus vulgaris) Bean root (P. vulgaris) Vetch root exudate(Vicia sativa sub sp. nigra) Compounds Luteolin Geraldone Genistein, Daidzein Delphinidin, Petunidin Naringenin Naringenin, Liquiritigenin

In some strains of R. meliloti, as many as 15 nodulation genes have been identified. Some of them are barely expressed in R. meliloti in a free-living state but are induced to express themselves manifold in the presence of root exudates. In alfalfa (lucerne, Medicago sativa), the induction factor in the root exudate is a flavone known as luteolin (3',4',5', 7'-tetrahydroxy flavanone). During the initiation of symbiosis, the two symbionts (the Rhizobium and the legume) interact at the molecular level of gene expression by signalling to each other. The product of nod D gene in Rhizobium interacts with the flavonoid compounds secreted by the roots and this follows the activation of other nod genes in a host dependent manner. Several flavonoid molecules produced by legumes have been found to induce or block the transcription of nod genes in Rhizobium and Bradyrhizobium. A schematic diagram of events at the molecular level in the root region in Rhizobiumlegume interaction. The protein product of nod D gene in Rhizobium is activated by

contact with plant signal by way of flavonoid compounds. This then interacts on the promoter gene which controls nod ABC genes action to induce nodule formation

The active inducers in root exudates of clover are 7,4-dihydroxyflavone, umbelliferone and formononetin, and soybean rootsrelease naringenin, genistein and diadzein (isoflavones). The positive action of nod genes in R. leguminosarum biovar trifolii is mediated by nod D gene and the interaction of nod D gene product with the plant secreted inducer and anti-inducer compounds. There are nodule inducer as well as anti-inducer regions on the clover root, the plant cells behind the root tip zone being the major site for the secretion of the inducer compounds. This region is followed upwards with regions on the root alternatively producing stimulatory or inhibitory substances. Apparently, the primary determining factor for sites of nodule initiation in clover and other legumes could be the ratio of stimulator (inducer): inhibitor (anti-inducer) in the vicinity of potential infection sites or the infection thread during infection. The anti-inducers are coumarins and isoflavones. Three components are necessary for induction of nod genes-1) the nod promoter, (2) the inducing substance produced by the plant which is of flavonoid nature, and 3) the nod D gene product. Some reports indicate that adding flavonoids (10 mm of luteolin or naringenin) to the root region of certain varieties of alfalfa (Medicago sativa) increased nodulation and N2 fixation by R. melitoli under controlled defined experimental conditions. Experiments have also

shown that certain flavonoids could be extracted from soils and are also present in roots of non-legumes such as wheat. It is too early to say whether these flavonoids present in soil and wheat root could really have a role in root nodulation, but the findings assume significance when we are aware that nitrogen fixing nodules could be induced on wheat and rape seedlings by inoculation with specific rhizobia in the presence of hydrolytic enzymes. The regulation of common nod genes in Bradyrhizobium japonicum appears to be similar to that of R. meliloti-alfalfasymbiosis. A positive activator nod D1 interacts with flavonoid compounds (daidzein and genestein) resulting in the induction of common nod genes. In R. meliloti a 14 Kb nif/fix gene cluster with essentially three symbiotically transcription units, have been delineated. These include the structural genes for nitrogenase (nif HDK), a regulatory gene (nif A), and genes of unspecified function (fx ABC). The development of nitrogen-fixing nodule involves the synthesis of a group of plant proteins, called nodulins. It has been demonstrated that there are nearly 100 nodulins involved in nodule development. Leghaemoglobin is a nodulin gene product. In soybean, the nodulin gene family in the host responsible for the synthesis of leghaemoglobin is activated 7-8 days after infection. Other enzymes in the nodules, the uricase and glutamine synthetase are also controlled by nodulins.

During the Rhizobium-legume symbiosis, bacteria enter the cells of host plants and differentiate into nitrogen-fixing bacteroids. Recent mutant screens and expression studies have revealed bacterial genes involved in the developmental pathway and demonstrate how the genetic requirements can vary from one host-microbe system to another. Multiplied bacteria cell remains dormant inside the plant cell- called bacteriods.

Unit IV: Agrobacterium and Plant viruses. Crown gall disease: A.tumefaciens causes crown gall disease. Tumerous growth in the shoots part of plants.-due to Ti plasmid that codes for the secretion of growth harmones like Auxins and cytokinin.

Genes involved in Crown gall disease: Oncogenes: 3 types of genes are involved 1.aux A genes- encodes-Tryptophan monooxygenase 2.aux B genes- encodes- Indole acetamide hydrolase. 3.Cyt gene encode-Isopentenyl transferase- catalyse in cytokinin production. Ti-Plasmid: Tumour inducing plasmid. Large ,Double stranded DNA present in A.tumefaciens. Transferred to plant cells through infection. Size-200 kb . It contains the following genes 1.Tumor inducing genes-T-DNA. 2.Vir genes 3.Ori 4.Occ 5.Tra 6.Nos or ocs. 7.Left and Right Border sequences. Ti Plasmid 2 Types 1.Octapine Ti plasmid 2.Nopaline Ti plasmid

Ti Plasmid derived vectors: 2types 1.Disarmed Ti plasmid 2.Binarry vectors. T-DNA and its Importance in genetic engineering. T-DNA consist of

1.Border sequence 2.T-DNA 3.Oncogenes. 4.tm1-gene 5.Genes for the production of Opines.

Importance: 1.used for cloning foreign genes in the T-DNA of Ti-plasmid.

Methods of gene transfer 1. Electroporation 2. Shot Gun method 3. Agrobacterium Mediated Gene Transfer -

The appropriate gene construct is inserted within the T-region of a disarmed Ti plasmid; either a cointegrate or a binary vector is used. The recombinant DNA is placed into Agrobacterium, which is co-cultured with the plant cells or tissues to be transformed for about 2 days. In case of many plant species, small leaf discs are excised from surface sterilized leaves and used for co cultivation.

In general, the transgene construct includes a selectable reporter gene, e.g., the bacterial neo gene. The neo gene is linked with suitable regulatory sequences that are functional in plant cells. During the leaf disc-Agrobacterium co-culture, acetosyningone released from plant cells induces the vir genes, which bring about the transfer of recombinant T-DNA into many of the plant cells. The T-DNA would become integrated into the plant genome, and the transgene would be expressed. As a result, the transformed plant cells would become resistant to kanamycin (due to the expression of neo gene). After 2 days, the leaf discs are transferred onto a regeneration medium containing appropriate concentrations of kanamycin and carbenicillin. Kanamycin allows only transformed plant cells to divide and regenerate shoots in about 3-4 weeks, while carbenicillin kills Agrobacterium cells: The shoots are separated, rooted and finally transferred-into soil. The use of leaf disc for co-culture is better than that of protoplasts or cultured cells since they are likely to show somaclonal variation. Efficient transformation of monocot cells can be obtained by providing acetosyringone during the co-culture of plant cells with Agrobacterium. Some plant species may secrete compounds, which inhibit the induction of vir operons by acetosyringone. This problem can be overcome by addition of an excess acetosyringone. Using this approach, successful transformation has been obtained in barley, wheat, maize and rice.

Features of Co integrate and Binary Vectors of pTi -

Vector type Sequences present Derived by Cointegrate 1. Single plasmid vector

Binary vector

2. pTi sequences: virregion, at least TDNA borders and origin of DNA replication + anE. coli plasmid, e.g., pBR 322, and some T-DNA sequences, including the DNA insert, placed within T-DNA (without borders). 1. A pair of Mini Ti produced plasmids; one by integrating pTi contains vir region, and the other has TDNA 2. One plasmid (called mini-Ti or micro-Ti is an E. coli plasmid, e.g., pRK252, which contains disarmed T-DNA; used for cloning of DNA insert in E. coli 3. A 'helper' pTi, which contains the T-DNA into an E. coli plasmid

Used in Example Transformation Homologous Used alone since it pGV2260 recombination has both vir region between an IV and T-DNA borders (used for cloning the DNA insert in E. coli) and a pTi (ordinarily disarmed)

Mini-Ti and 'helper' pTi must be used together; both the plasmids must be present in the same Agrobacterium cell.

Bin19 (Mini-Ti) and pAL 4404 (helper Ti)

Helper obtained

pTi by

vir region, but deletion of T-DNA ordinarily lacks T- sequences from pTi DNA (including the border sequences) Plant Viral Vectors: Plant virus used as viral vectors include 1.Gemini virus. 2.Cauliflower virus

Cauliflower mosaic virus: Belongs to Caulimovirus group of plant viruses. Infect members of family cruciferae. Transmitted Aphids. DNA OF CaMv: Circular ,double stranded . 8024bp Has 3 gaps circular DNA-at specific sites. Consist of 6 closely packed genes and a small intergenic sequence. Gene-1- spreading of virus. Gene -2 Aphid mediated dispersal of virus. Gene 3 &4-structural proteins of virus capsid. Gene -5-Enryme reverse transcriptase. Gene-6-Protein Viroplasm.

CaMv As a Vector: Cauliflower mosaic virus genome

Intergenic sequence cleaved Foreign DNA- cloned Resulting in recombinant CaMv virus. Gently rubbed on leaf surface virus particle reproduce-inside plant cells. Replication: Double stranded DNA transcription-RNA Polymerase synthesis 35s mRNA-codes for viral coat proteins and by using reverse transcriptase synthesis viral DNAget assembles in viral coat proteins and infect other cells. Gemini virus Paired particle morphology. Covalently closed circular single stranded DNA. DNA -2.6-3.0 kb long. Converted to double stranded replicative form upon replication.

Example

Bean golden mosaic virus.(BGMV) 2510bp long Genome is bipartite Has DNA A and DNA-B.

DNA A- Codes proteins responsiable for replication. Gemini virus is of two types Gemini virus infecting Monocot-eg-Maize streak virus. Gemini virus infecting Dicot-Cassia virus. Use of Gemini virus: Genes coding for vp protein is replaced by foreign proteins. 3kb of foreign DNA can be cloned. Disadvantages: Not readily transformed by mechanical means.

Viral vectors have been proven to be the most efficient in gene delivery, leading to more than 30,000 publications since 1990. The extensive use of viral vector techniques has become an expressway to the target of functional genomics. Many viral vector systems have been developed over time and adenovirus, adeno-associated virus (AAV), and retrovirus become widely used. Among the 1000 gene therapy clinical trails worldwide, more than 55% use these three type of vectors.The reasons for their popularity are as below: 1) Better understanding of the virology. These viral systems are well studied. Significant amount of knowledge on the viral life cycle as well as viral protein functions are accumulated during academic research. This information allows construction of recombinant viral vector to hold non-viral genes for delivery, as well as complimentary systems to produce the recombinant (usually defective) virus. 2) Established application in gene delivery. Again, cumulative data on the use of these viral vectors in gene delivery to various cells and tissues help other researchers to make their choices. People need to know the vector tropism, infection efficiency, immune response, and potential toxicity before designing their own experiments. 3) Significant advantage in performance. Practical reasons such as ease of construction, good production titer, high transgene expression, and high safety profiles determine the application potential of the viral vectors. Adenovirus, AAV, and retrovirus are the most commonly used viral systems due to the established protocol, excellent performance in gene transduction, and relatively safe to use in standard lab setting.

Gene Delivery Techniques So what kind of methods are out there for gene delivery? For in vitro studies, plasmid transfection is commonly used. It is easy to perform, fast, and can be used for transient expression or generating stable cell lines. However, this non-viral based method has highly variable efficiency depending on cell types. Most of the time <10% of the cells can be transfected even with the powerful lipid-based DNA complex. Also, the efficiency for establishing genome- integrated, stable cell line is much lower (<0.01%). The biggest advantage of using viral vectors for in vitro studies is high efficiency. Viral vectors can infect 100% of the cells at the adequate titer. Adeno, AAV, and retroviruses all have very broad tropism, and are capable of infecting all kind of cells with high efficiency. When your study requires the whole cell population to be transduced (such as gene downregulation by RNAi), or your work involves some "difficult" cell lines which are refractory to plasmid transfection, viral vectors are your best choice. In vivo gene delivery is dominated by viral vector approaches. Viral vectors can delivered by intravenous infusion or local injection. Since viral infection is receptor mediated, tissue specificity of viral vectors can be achieved by its specific tropism, through pseudotyping, or capsid modification. The use of tissue specific promoters also help to limit the recombinant gene expression in target tissues. The high efficiency in gene delivery makes possible both in vivo gene function research as well as gene therapy studies for certain diseases. With many different viral vectors systems available, researchers can make the best choice based on the inherent properties of each system for their particular applications.

UNIT V

APPLICATION OF PLANT BIOTECHNOLOGY

Transgenic plants: Transgenic plants are plants that have been genetically engineered, a breeding approach that uses recombinant DNA techniques to create plants with new characteristics.They are identified as a class of genetically modified organism (GMO).

Pest resistant plants : Plants showing resistance to attack by insect pests called-Pest resistant plants. Insect attack is a serious agricultural problem leading to yield losses and reduced product quality. Insects can cause damage both in the field and during storage in silos. Each year, insects destroy about 25 percent of food crops worldwide. The larvae of Ostrinia nubilalis, the European corn borer, can destroy up to 20 percent of a maize crop. The Bt concept pest resistant transgenic plants Bacillus thuringiensis, or Bt, is a bacterium that has attracted much attention for its use in pest control. The soil bacterium produces a protein that is toxic to various herbivorous insects. The protein, known as Bt toxin, is produced in an inactive, crystalline form. When consumed by insects, the protein is converted to its active, toxic form (delta endotoxin), which in turn destroys the gut of the insect. Bt preparations are commonly used in organic agriculture to control insects, as Bt toxin occurs naturally and is completely safe for humans. More than 100 different variations of Bt toxin have been identified in diverse strains of Bacillus thuringiensis. The different variations have different target insect specificity. For example, the toxins classified under Cry1a group target Lepidoptera (butterflies), while toxins in the Cry3 group are effective against beetles. Researchers have used genetic engineering to take the bacterial genes needed to produce Bt toxins and introduce them into plants. If plants produce Bt toxin on their own, they can defend themselves against specific types of insects. This means farmers no longer have to use chemical insecticides to control certain insect problems.

Critics claim that in some cases the use of insect resistant crops can harm beneficial insects and other non-target organisms. Extensive ecological impact assessments have been addressing these issues. In the field, no significant adverse effects on nontarget wildlife nor long term effects of higher Bt concentrations in soil have yet been observed. New concepts on the way Bt crops have been planted commercially for more than eight years. Other naturally occuring insecticidal compounds are now becoming available as alternatives to the Bt approach. Among these are chitinase, lectins, alpha-amylase inhibitors, proteinase inhibitors, and cystatin. Plants genetically modified to express these defense proteins are still in early stages of development. Bacillus thuringiensis : Gram negative bacteria Produce delta toxin. Plasmid carring Cry genes codes for delta toxin insecticidal activity. Toxin consist of 3 domins . Domain 1 responsiable for creating pores in the insect guts. Domain 2-Receptor recognition. Domain 3- Protection from degradation,Toxin bilayer interaction. Genes: Cry 1Aa active against Lepidoptera. Cry2Aa- active against Lepidoptera and Dipteria. Cry3Aa Coleopteria.

Herbicide resistant plants :

Herbicide resistant crops Excessive weed growth forces crops to compete for sunlight and nutrients, often leading to significant losses. Because herbicides cannot differentiate between plants that are crops and plants that are weeds, conventional agricultural systems can only use 'selective' herbicides. Such herbicides do not harm the crop, but are not effective at removing all types of weeds. If farmers use herbicide resistant crops, 'non-selective' herbicides can be used to remove all weeds in a single, quick application. This means less spraying, less traffic on the field, and lower operating costs.

'Non-selective' herbicides: Not always useful 'Broad-spectrum', or non-selective herbicides are effective at killing a wide range of weeds. The problem is, they can also kill valuable crops. Therefore, broad-spectrum herbicides are only useful before seedlings emerge or in special cases like fruit orchards, vineyards, and tree nurseries. Herbicide resistant crops are changing weed managment Several crops have been genetically modified to be resistant to non-selective herbicides. These transgenic crops contain genes that enable them to degrade the active ingredient in an herbicide, rendering it harmless. Farmers can thereby easily control weeds during the entire growing season and have more flexibility in choosing times for spraying. Herbicide resistant crops also facilitate low or no tillage cultural practices, which many consider to be more sustainable. Another advantage is that farmers can manage weeds without turning to some of the more environmentally suspect types of herbicides. Critics claim that in some cases, the use of herbicide resistant crops can lead to an increase in herbicide use, promote the development of herbicide resistant weeds, and damage biodiversity on the farm. Extensive ecological impact assessments have been addressing these issues. Among the field trials conducted on herbicide resistant crops, studies in the United Kingdom have shown that different herbicides and different herbicide application practices can affect the amount of wild plants on the farm. In comparison with conventional cropping systems, weed and animal populations were negatively affected by herbicide tolerant sugar beet and rapeseed, but biodiversity was increased with the use of herbicide tolerant maize. Currently, two herbicide resistant cropping systems are common for soybean, maize, rapeseed, and cotton: RoundupReady (active agent: glyphosate) and Liberty Link (active agent: glufosinate). Herbicides are chemicals that specically inhibit the plant by interfering the synthesis of essential Proteins. Stratergies for engineering herbiside resistance plants: 1.Overexpression of the target Protein. Petunia cDNA of ESPS gene from glyphosate resistant plants and introduced into sensitive plants. 2.Mutation of target protein.

Mutant EPSPS gene isolated from glyphosate resistant bacteria - transfer to sensitive plants.

3.Detoxification of the herbicide using a Single gene from a Foreign source. GOX gene from O.anthropi isolated and modified and transferred into oilseed raps-good glyphosate resistance. 4.Enhancing plant detoxification. Drought resistant and cold resistant plants Drought tolerance refers to the degree to which a plant is adapted to arid or drought conditions. Desiccation tolerance is an extreme degree of drought tolerance. Plants naturally adapted to dry conditions are called xerophytes. Drought tolerant plants typically make use of either C4 carbon fixation or crassulacean acid metabolism (CAM) to fix carbon during photosynthesis. Both are improvements over the more common but more basal C3 pathway in that they are more energy efficient. CAM is particularly good for arid conditions because carbon dioxide can be taken up at night, allowing the stomata to stay closed during the heat of day and thus reducing water loss. Many adaptations for dry conditions are structural, including the following:

Adaptations of the stomata to reduce water loss, such as reduced numbers or waxy surfaces. Water storage in succulent above-ground parts or water-filled tubers. Adaptations in the root system to increase water absorption. Trichomes (small hairs) on the leaves to absorb atmospheric water.

Importance in agriculture
Arid conditions can lower the yield of many crops. Plant breeding programs for improved yield during drought conditions have great economic importance, and these programs may be broad in scope. For example, one study on soybeans currently being conducted by the United States Department of Agriculture is scheduled to span several years, with research taking place across that country, and has among its goals the identification of specific mechanism by which soybeans resist wilting and of the specific genes for drought tolerance.

Importance in horticulture
In landscapes in arid or drought-prone regions, drought tolerance is an important consideration in plant selection. Xeriscaping is an approach to landscaping first developed in Denver, Colorado, a region with hot, dry summers. The use of drought tolerant plants is essential to a successful xeriscape, which ideally requires no supplemental irrigation.

Salt and stress tolerance plants Halophytes are salt-tolerant higher plants. naturally halotolerant plants or microorganisms could be developed into useful agricultural crops. Tolerance of high salt conditions can be obtained through several routes. High levels of salt entering the plant can trigger ionic imbalances which cause complications in respiration and photosynthesis, leading to reduced rates of growth, injury and death in severe cases. To be considered tolerant of saline conditions, the protoplast must show methods of balancing the toxic and osmotic effects of the increased salt concentrations. Halophytic vascular plants can survive on soils with salt concentrations around 6%, or up to 20% in extreme cases. Tolerance of such conditions is reached through the use of stress proteins and compatible cytoplasm osmotic solutes. To exist in such conditions, halophytes tend to be subject to the uptake of high levels of salt into their cells, and this is often required to maintain an osmotic potential lower than that of the soil to ensure water uptake. High salt concentrations within the cell can be damaging to sensitive organelles such as the chloroplast, so sequestration of salt is seen. Under this action, salt is stored within the vacuole to protect such delicate areas. If high salt concentrations are seen within the vacuole, a high concentration gradient will be established between the vacuole and the cytoplasm, leading to high levels of energy investment to maintain this state. Therefore, the accumulation of compatible cytoplasmic osmotic solutes can be seen to prevent this situation from occurring. Amino Acids such as proline accumulate in halophytic Brassica species, quaternary ammonium bases such as Glycine Betaine and sugars have been shown to act in this role within halophytic members of Chenopodiaceae and members of Asteraceae show the build up of cyclites and soluble sugars. The build up of these compounds allow for the balancing of the osmotic effect while preventing the establishment of toxic concentrations of salt or requiring the maintenance of high concentration gradients.

Contaminant tolerant plants that can grow in the presence of high levels of toxic compounds and accumulate them in their tissues The removal from the environment of many potentially toxic compounds is complicated by the numerous classes and types of these chemicals. For example, many soils are contaminated with one or more metals, radioactive or inorganic compounds. Of these, the metals may include lead, zinc, cadmium, selenium, chromium, cobalt, copper, nickel and mercury; the radioactive compounds may be uranium, cesium or strontium; and the other inorganic compounds might include arsenic, sodium, nitrate, ammonia or phosphate. Soil may become polluted with high concentrations of metals by either a natural phenomenon such as proximity to an ore body, or as a consequence of industrial activities. The remediation of heavily metal-contaminated soils often involves excavation and removal of soil to "secured" landfills, a technology that is expensive and requires site restoration . As an alternative, in the past few years, several groups of scientists have begun to develop technological approaches to using certain plants to remove metal contaminants from the soil. Phytoremediation is a relatively new approach to removing contaminants from the environment. It may be defined as the use of plants to remove, destroy or sequester hazardous substances from the environment. Unfortunately, even plants that are relatively tolerant of various environmental contaminants often remain small in the presence of the contaminant . Genetic modification of plants has been useful in bioremediation. Some plants have been specially bio-engineered to enable them remove toxic waste from the environment. Several researchers have reported encouraging results using plants like mustard greens, alfalfa, river reeds, poplar trees, and special weeds to clean up the ravages of industries, agriculture, and petroleum production In some cases, plants can digest the poisons, and convert them to inert compounds .

Tomato plant genes used to increase metal stress tolerance of Canola plants for phytoremediation . It was previously observed that transgenic tomato plants that express the Enterobacter cloacae UW4 1-aminocyclopropane-1-carboxylate (ACC) deaminase (EC 4.1.99.4) gene, and thereby produce lower levels of ethylene, were partially protected from the deleterious effects of six different metals. However, since tomato plants are unlikely to be utilized in the phytoremediation of contaminated terrestrial sites, transgenic canola (Brassica napus) plants

that constitutively express the same gene were generated and tested for their ability to proliferate in the presence of high levels of arsenate in the soil and to accumulate it in plant tissues. The ability of the plant growth-promoting bacterium E. cloacae CAL2 to facilitate the growth of both non-transformed and ACC deaminase-expressing canola plants was also tested. In the presence of arsenate, in both the presence and absence of the added plant growth-promoting bacterium, transgenic canola plants grew to a significantly greater extent than non-transformed canola plants.

Molecular pharming and theraputic products. Plant molecular farming uses genetic engineering to produce substances for a variety of uses. Potential products include the development of antigens for vaccines that might be massproduced in plants such as corn and used to fight such diseases as cancer and diabetes.

Advantages Plants do not carry pathogens that might be dangerous to human health. Additionally, on the level of pharmacologically active proteins, there are no proteins in plants that are similar to human proteins. On the other hand, plants are still sufficiently closely related to animals and humans that they are able to correctly process and configure both animal and human proteins. Their seeds and fruits also provide sterile packaging containers for the valuable therapeutics and guarantee a certain storage life. Global demand for pharmaceuticals is at unprecedented levels, and current production capacity will soon be overwhelmed. Expanding the existing microbial systems, although feasible for some therapeutic products, is not a satisfactory option on several grounds. First, it would be very expensive for the pharmaceutical companies. Second, other proteins of interest are too complex to be made by microbial systems. These proteins are currently being

produced in animal cell cultures, but the resulting product is often prohibitively expensive for many patients. Finally, although it is theoretically possible to synthesize protein molecules by machine, this works only for very small molecules, less than 30 amino acid residue in length. Virtually all proteins of therapeutic value are larger than this and require live cells to produce them. For these reasons, science has been exploring other options for producing proteins of therapeutic value.

Molecular pharming includes Pharming of carbohydrates Amylase Cyclodextrins Frucyans Amylopectin. . Fats Saturated fatty acids. Mono unsaturated fattyacids Medium chain fattyacids PHB. Transgenic proteins. Antibodies Subunit vaccines Enzymes Hirudin Hman serum Albumin.

Outline of plant tissue culture: Growing plant cells under invitro-Plant tissue culture. Tissue culture is of two types 1.Direct embryogenesis.

From the source culture direct organogenesis takesplace. 2.Indirect embryogenesis: From the source culture an intermediate callus is formed ,from the callus the organogenesis takes place.