BT 6010 - Plant Department :Biotechnology 2018- 19

DEPARTMENT OF BIOTECHNOLOGY

Faculty Name : Dr. A. Sai Ramesh

Faculty Code : HTS 1396

Subject Name : PLANT BIOTECHNOLOGY

Subject Code : BT6010 (C321)

Year & Semester : III & VI

Date of Compilation : 22/11/18

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19

DEPARTMENT OF BIOTECHNOLOGY COURSE DETAILS

Faculty Name : Dr. A. Sai Ramesh Faculty Code: HTS 1396 Subject Name: PLANT BIOTECHNOLOGY Subject Code: BT6010 (C321) Department: Biotechnology Year & Semester: III & VI Date of Compilation: 22/11/18

COURSE OBJECTIVES

Course Outcomes: At the end of the course, the student will be able to:

Knowledge CO No Course Outcomes Level C321.1 Understand the fundamentals of plant cells, structure and functions K2 C321.2 Understand the regulation and transport of proteins K2 C321.3 Learn the nitrogen fixation mechanism and significance of viral vectors K2 C321.4 Gain the knowledge about the plant and transgenic plants K3 C321.5 Use of the gained knowledge for the development of therapeutic products K3

Mapping of Course Outcomes with Program Outcomes

CO PO1 PO 2 PO 3 PO 4 PO 5 PO 6 PO 7 PO 8 PO 9 PO 10 PO 11 PO 12 C321.1 2 ------C321.2 2 ------C321.3 2 - 1 1 - 1 1 - - - 1 2 C321.4 2 1 3 2 - 1 1 1 - - 2 2 C321.5 2 2 3 3 - 2 3 2 - 1 2 2

C.No PO1 PO 2 PO 3 PO 4 PO 5 PO 6 PO 7 PO 8 PO 9 PO 10 PO 11 PO 12 C321 2 - 3 - - 1 1 - - - 2 2

Mapping Relevancy 3 – Substantial (Highly relevant), 2 – Moderate (Medium), 1 – Slight (Low)

Course delivery methods  Class room lecture - Black board  PPTs, Videos  Lab Demonstrations  Activities like In Plant Training, Industrial Visit and Guest Lecture

Assessment methods  Continuous Internal Assessment  Assignments  Seminars VTHT Page 2

BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19

BT6010 PLANT BIOTECHNOLOGY L T P C 3 0 0 3 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 in Plant and how to develop Transgenic plants. This will facilitate the student to take up project work in this area.

UNIT I ORGANIZATION OF GENETIC MATERIAL 9 Genetic material of plant cells – nucleosome structure and its biological significance; junk and repeat sequences; outline of transcription and translation.

UNIT II CHLOROPLAST & MITOCHONDRIA 9 Structure, function and genetic material; rubisco synthesis and assembly, coordination, regulation and transport of proteins. Mitochondria: Genome, cytoplasmic male sterility and import of proteins.

UNIT III NITROGEN FIXATION 9 Nitrogenase activity, nod genes, nif genes, bacteroids.

UNITIV & VIRAL VECTORS 9 Pathogenesis, crown gall disease, genes involved in the pathogenesis, Ti plasmid – t-DNA, importance in genetic engineering. Viral Vectors: Gemini virus, cauliflower mosaic virus, viral vectors and its benefits.

UNIT V APPLICATION OF PLANT BIOTECHNOLOGY 9 Outline of plant tissue culture, transgenic plants, and pest resistant plants, molecular , theraputic products. TOTAL: 45 PERIODS TEXT BOOKS: 1. Chawla, H.S., “Introduction to Plant Biotechnology”, 3rd Edition, Science Publishers, 2009. 2. Gamburg OL, Philips GC, Plant Tissue & Organ Culture fundamental Methods, Narosa Publications. 1995.

REFERENCES: 1. Stewart Jr., C.N., “Plant Biotechnology and : Principles, Techniques and Applications” Wiley-Interscience, 2008. 2. Heldt HW. Plant Biochemistry & Molecular , Oxford University Press. 1997. 3. Ignacimuthu .S, Applied Plant Biotechnology , Tata McGraw Hill. 1996.

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19

BT6010 PLANT BIOTECHNOLOGY

Unit I ORGANIZATION OF GENETIC MATERIAL

1. Differentiate prokaryotic and eukaryotic genetic material. In prokaryotes genetic material is present inside the nucleus and packed well by histones. In eukaryotes there is no nucleus and histones. 2. Write notes on nucleosome. Nucleosomes are bead like structures, each bead contain around 200 bp DNA, RNA and histonic and non histonic proteins. Histonic proteins are responsible for DNA condensation. 3. What are histone proteins? Explain their role.(Nov. 2011, May 2012, Nov. 2014, May. 2014, May - 16) These are basic proteins; 10 – 20 of aminoacids are basic aa’s like Arginine and Lysine. H2a, H2b, H3, H4 are involved in octomer formation. The condensation of eukaryotic DNA occurs only in the presence of histonic octomeres and the H1 proteins are playing major role in sealing the nucleosome structure. 4. Write notes on solenoid structure Interphase chromosome is 30 nm thick,it is formed due to the folding of nucleosome chain into a solenoid structure , each turn is having 6 nucleosomes and a central hole. 5. Explain the work of Watson and Crick DNA is made of double helical structure two right handed helical polynucleotide chain run opposite direction and two strands are held together by hydrogen bonds. 6. Enumerate the difference between B-DNA and Z-DNA. B-DNA is the right handed form, at 92% relative humidity 10bp/turn it is the most stable form. Z – DNA is the zig zag form and 12 bp/turn. 7. What is A-DNA? It is a slightly right handed form, at 62% relative humidity 11bp/turn and tilted 20 away from the helical axis. 8. What are plasmids? These are extrachromosamal self replicating double slandered, closed circular DNA Plasmids contains genes for autoreplication, restriction site, antibiotic resistance, metal resistance and Pollutant degradation, ex: Ti and Ri plasmid. 9. What do you mean by DNA replication? It is a process in which DNA multiplication occurs, parental strands will be acting as a template and new strands are complementary to the template strands, for this DNA polymerases, templates, nucleotide triphosphates, etc. are needed. 10. Differate rolling circle and theta mode of replication Rolling circle replication has a peculiarity that the new strand rolls over the template strand until the completion of the new strand synthesis. In theta mode the intermediate formed looks like a letter Thetta. 11. Write notes on Sigma factor. RNA polymerase in prokaryotes are made of five different sub units among this sigma factor is acting as an initiator for transcription, after transcribing a short segment of RNA this factor will be removed from the other factors. 12. Give two examples for gene regulation in plants? Etiolation in plants that is a dark plants lose their chlorophyll and their green colour. Expression of heat shock proteins in plants 13. What is sigma factor? Sigma factor is a subunit of RNA polymerase in prokaryotes, it is responsible for recognising the initiation site for transcription in prokaryotes, after initiation it will be relieved from the core enzyme. 14. Differentiate conservative and semiconservative replication

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 Conservative is a replication in which the parental strands will be retained and there is no denaturation occur. But in semiconservation denaturation of the double strand occur and each strand will be act as a template from which complementary strands will be synthesised. 15. What do you mean by a transcription unit? RNAs are transcribed in different units each unit is separated by non transcribed spacers these are repeating units. Ex in Xenopus 5.8, 18,28s rRNA are in a single unit. 16. Differentiate RNA polymerase in Pro and eukaryotes. In prokaryotes RNA polymerase contain 5 different sub units among this Sigma sub unit is responsible for the initiation of transcription. In eukaryotes 3 types of RNA polymerases are present each one is responsible for the synthesis of different rRNAs. RNA polymerase I - 5.8s,18s& 28s rRNAs, RNA polymerase II – tRNA &5s rRNA and RNA polymerase III – RNA. 17. Name few processing events in tRNA. Removal of introns in Yeast tRNA and addition of CCA sequence, etc. 18. What is the difference between transcription and translation? Transcription is the DNA dependant RNA synthesis, but translation is the protein synthesis. 19. What is aminoacylation of tRNA ? Aminoacylation is a process in which tRNA is added with aminoacid and the tRNA with the aminoacid is called as acylated tRNA. 20. Explain tRNAmet in E.coli. In E.coli the tRNA charged with initiation aminoacid methionine is formilated methionine it is designated as tRNA fmet. 21. Explain Shine Dalgarno sequence. During translation, binding of 30s ribosomal subunit on mRNA and 3’OH end of 16srRNA occur, this stretch of complementary sequence in mRNA with 16srRNA is called as Shine Dalgarno sequence. 22. What do you mean by translocation? When the peptide bond formation is over, the acylated tRNA from A-site moved into the P-site this process is called as translocation. 23. What is peptidyl transferase centre? Peptide bond formation between aminoacids in A and P site occur in the prescence of an enzyme peptidyl transferase, this reaction occur in the 50s ribosomal subunit hence it is called as peptidyl transferase centre. 24. What are polysomes? In prokaryotes during polypeptide chain elongation number of ribosomes will be attached on a single mRNA this cluster of ribosomes on mRNA is called polysomes. 25. Differentiate eukaryotic and prokaryotic mRNA. In prokaryotes it is polycistronic but in eukaryotes it is monocistronic and in eukaryotes 5’ capping and poly-A tail will be there. 26. List out some properties of genetic material? (May 2012). For a molecule to serve as the genetic material, it must be able to replicate, store information, express information, and allow variation by mutation. The genetic material is physically transmitted from parent to offspring. Proteins and nucleic acids were the major candidates for the genetic material. 27. Mention any two advantages of junk DNA? (May 2012, May. 2014, Nov. 2014 May – 16). Many noncoding DNA sequences have important biological functions as indicated by comparative genomics studies that report some regions of noncoding DNA that 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. Linkage mapping often identifies chromosomal regions associated with a disease with no evidence of functional coding variants of genes within the region, suggesting that disease-causing genetic variants lie in the noncoding DNA. Some specific sequences of noncoding DNA may be features essential to chromosome structure, centromere function and homolog recognition in meiosis. 28. What are the different compartments in which DNA is found in plant cells and name one gene from each compartment? (Nov. 2014)  Nucleus – gene for small Rubisco subunit.

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19  Chloroplast – gene for large Rubisco subunit.  Mitochondria – gene for male sterility.

29. How does methyl jasmonate elucidate transcription control? (May. 2015) Jasmonates (JAs) are plant-specific signaling molecules that steer a diverse set of physiological and developmental processes. Pathogen attack and wounding inflicted by herbivores induce the biosynthesis of these hormones, triggering defense responses both locally and systemically. We report on alterations in the transcriptome of a fast-dividing of the model plant after exogenous application of methyl JA (MeJA). Early MeJA response genes encoded the JA biosynthesis pathway proteins and key regulators of MeJA responses, including most JA ZIM domain proteins and MYC2, together with transcriptional regulators with potential, but yet unknown, functions in MeJA signaling. In a second transcriptional wave, MeJA reprogrammed cellular metabolism and cell cycle progression. Up-regulation of the monolignol biosynthesis gene set resulted in an increased production of monolignols and oligolignols, the building blocks of lignin. Simultaneously, MeJA repressed activation of M-phase genes, arresting the cell cycle in G(2). MeJA- responsive transcription factors were screened for their involvement in early signaling events, in particular the regulation of JA biosynthesis. Parallel screens based on yeast one- and transient transactivation assays identified both positive (MYC2 and the AP2/ERF factor ORA47) and negative (the C2H2 Zn finger proteins STZ/ZAT10 and AZF2) regulators, revealing a complex control of the JA autoregulatory loop and possibly other MeJA-mediated downstream processes. 30. Describe the eukaryotic ribosome structure and its active centers? (Nov. 2012). Ribosomes consist of two major subunits—the small ribosomal subunit reads the mRNA, while the large subunit joins amino acids to form a polypeptide chain. Each subunit is composed of one or more ribosomal RNA (rRNA) molecules and a variety of proteins.

31. What are nucleosomes? How do they help in gene regulation? (May. 2015) Mutilation of DNA and histone causes nucleosomes to pack tightly together. Trascription factors cannot bind the DNA and genes are not expressed.

Histone acetylation results in loose packing of nucleosomes. Transcription factors can bind the DNA and genes are expressed.

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19

32 .Define chromatin.( May2017) Chromatin is a complex of macromolecules found in cells, consisting of DNA, protein, and RNA.[1] The primary functions of chromatin are 1) to package DNA into a more compact, denser shape, 2) to reinforce the DNA macromolecule to allow mitosis, 3) to prevent DNA damage, and 4) to control gene expression and DNA replication. The primary protein components of chromatin are histones that compact the DNA. Chromatin is only found in eukaryotic cells (cells with defined nuclei). Prokaryotic cells have a different organization of their DNA (the prokaryotic chromosome equivalent is called genophore and is localized within the nucleoid region). 33. State the significance of junk DNA. (May2017) Some of this noncoding DNA is used to produce noncoding RNA components such as transfer RNA, regulatory RNA and ribosomal RNA. However, other regions are not transcribed into proteins, nor are they used to produce RNA molecules and their function is unknown.

Part- B 1. Write notes on the condensation of Eukaryotic DNA.(Nov/Dec2013) 2. What do you mean by replication, explain it neatly? 3. Write notes on i) Nucleosome ii) Types of DNA 4. Explain in detail on i). Cot analysis ii). mRNA splicing 5. Write notes on the Eukaryotic translation and post translational modifications. 6. Give a detail account on the translation mechanisms of eukaryotic system .(May-14) 7. What is transcription, explain Eukaryotic transcription in detail. (May2017) 8. Write a detailed notes on RNA splicing. 9. Differentiate post transcriptional and tanslational modifications in detail. 10. What are the three types of RNA? Describe their structure and role in translation. (Nov. 2011). 11. Give a detailed account on transcription initiation, elongation and termination of a eukaryotic gene? (May 2012, Nov/Dec2013) 12. Explain complex organization of genetic material in a plant cell with illustration. (May 2012). 13. Elaborate on the nucleosome structure and organization of eukaryotic DNA? (Nov 2012, May- 16). 14. Elaborately discuss about the structural organization of nucleosome and its dynamics. (May 2014) 15. Explain the various post- transcriptional and translational modifications that regulate gene expression in detail. (Nov 2012, Dec2013). 16. Elaborate the various levels of gene regulation in eukaryotic protiein coding genes. (Nov 2014) 17. Draw the structure of a eukaryotic gene and promoter structure and explain the stage of transcription. (Nov. 2014) 18. Discuss in detail the plant genome organization. How would you differentiate it from prokaryotic system?. (May. 2015, May2017) 19. Enumerate and describe the transcription factors that play a key role in plant gene expression? (May. 2015) 20. Write detailed notes on junk and repeated sequence with neat sketch.

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19

Unit II CHLOROPLAST & MITOCHONDRIA

1. Differentiate chloroplast DNA and genomic DNA. Chloroplast DNA is circular, covalently closed and very similar to Prokaryotic DNA but the genomic DNA is linear, double stranded and contain nucleosome. 2. What do you mean by photophosphorylation? It is the process in which energy rich compounds are produced in the presence of light. ex. Cyclic and non- cyclic photophosphorylation. 3. Write notes on cyclic photophosphorylation. In this phosphorylation process electrons are cycled again and again passing via many different electron carriers. In each cycle one ATP is produced. 4. What do you mean by non-cyclic photophosphorylation? In this process electrons are generated through photolysis of water and electrons once produced will not be reutilised. 5. What are CAM plants? Crassulacean Acid Metabolism is observed in some plants called as CAM plants ex., Vitaceae,Orchidaceae, etc. 6. Explain lac operon. Operators are regulated by the presence or absence of lactose called as Lac operon .Lactose act as an inducer in E.Coli, in presence of lactose this will switch on the structural genes for the synthesis of B- galactosidase, permease and transacetylase responsible for the lactose catabolism. In the absence of lactose this gene will be switched off. 7. What are the upstream activation sites? In eukaryotes lengthy sequences occur hundreds of basepairs upstream from the transcription start site, which control the rate of initiation called upstream activation sites 8. Write notes on TATA box. In eukaryotes promoter region 25 basepair upstream from the start site consensus sequence TATAAT us there called as TATA or Hogness box, it is flanked by GC rich sequnce. 9. What is cat box? In many eukaryotes promoters posses consensus sequence like GG [T/C] CAATTCT called CATT box. 10. What are enhancers? It is a sequence, its location can be changed without altering its activity called enhancers,Identified in SV-40 virus, deletion of this region will reduce the transcriptional activity of the related gene 100 fold. 11. What is RNA splicing? In eukaryotes primary transcript undergo processing tobecome a functional form called as splicing ex. Removal of introns. 12. Write notes on protein trafficking (Nov. 2014). Protein synthesis occur on the surface of ER membrane, the newly synthesized proteins translocated from the site of synthesis to the site of activity through the barriers like inner and outer membrane and the space between membrane is called as Protein trafficking. 13. Name few post translational modifications. Removal of methionine, acetylation of N-terminal aminoacid, hydroxylation of proline and lysine, glycosylation,etc. 14. What is the localization signal for the protein resident in the lumen of the E.R? Addition of short aminoacid sequence at C-terminal end a tetrapeptide like KDEL is added, in yeast HDEL or DDEL is added. 15. What do you mean by cytoplasmic male sterility?(May - 16)(may-17) It is a phenomenon involved in mitochondrial genome. It is exploited by the breeders for producing hybrid lines of crops ex.Maize.

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 16. What are the advantages of cytoplasmic male sterility? It eliminate the need for mechanical or hand emasculation.Gene transfer occur through egg not through pollen. 17. What are the salient features of chloroplast genome? (Nov. 2011). 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.

18. What do you mean by power house of cell? (May 2012). Mitochondria is the powerhouse of the cell, which produces most of the ATP of the cell. 19. What are the three compartments in cell containing DNA and mention their origin? (Nov 2012). Nucleus, chloroplast and mitochondria. 20. Differentiate cytoplasmic and gene male sterility? (Nov 2012). Cytoplasmic male sterility is maternal and gene male sterility is paternal. cytoplasmic male sterility is due to the influence of gene that present in the mitochondrial plasmid but gene male sterility due to the genes present in the chromosome. 21. Draw the structure of chloroplast and comment on its salient features. (May. 2014)

22. Write about the structural organization of mitochondrial DNA. (May. 2014) Although most DNA is packaged in chromosomes within the nucleus, mitochondria also have a small amount of their own DNA. This genetic material is known as mitochondrial DNA or mtDNA. Mitochondrial DNA contains 37 genes, all of which are essential for normal mitochondrial function. Thirteen of these genes provide instructions for making enzymes involved in oxidative phosphorylation. The remaining genes provide instructions for making molecules called transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), which are chemical cousins of DNA. These types of RNA help assemble protein building blocks (amino acids) into functioning proteins. 23. What is the role of transit peptide in plant system? (May. 2015) The role of the transit peptide in the routing of imported proteins inside the chloroplast was investigated with chimeric proteins in which the transit peptides for the nuclear-encoded ferredoxin and plastocyanin precursors were exchanged. Import and localization experiments with a reconstituted chloroplast system show that the ferredoxin transit peptide directs mature plastocyanin away from its correct location, the thylakoid lumen, to the stroma. With the plastocyanin transit peptide-mature ferredoxin chimera, a processing intermediate is arrested on its way to the lumen. We propose a two domain hypothesis for the plastocyanin transit peptide: the first domain functions in the chloroplast import process, whereas the second is responsible for transport across the thylakoid membrane. Thus, the transit peptide not only targets proteins to the chloroplast, but also is a major determinant in their subsequent localization within the organelle. 24. What is male sterility? How is it applied to ? (May. 2015) Male sterility is defined as an absence or non-function of pollen grain in plant or incapability of plants to produce or release functional pollen grains. The use of male sterility in hybrid seed production has a great importance as it eliminate the process of mechanical emasculation. Types of Male Sterility:

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 The male sterility is of five types 1) Genetic male sterility, 2) Cytoplasmic male sterility, 3) Cytoplasmic genetic male sterility, 4) Chemical induced male sterility and 5) Transgenic male sterility. 25. Comment the significance of chloroplast? (May - 16) Chloroplasts are the source of virtually all of the world's food and fuel and much of its oxygen supply, and as such life on Earth depends on them. They are a vital component of all photosynthetic cells in plants and algae, and are unique to them. What makes them so important is that they are the sites of photosynthesis, from the absorption of light by chlorophyll through to the production of the first simple sugars. Chloroplasts are important in making other essential plant products, such as fats, oils, scents, and proteins. They can even make many of the proteins needed to produce another chloroplast. They are thought to have been originally free-living, single-celled photosynthetic bacteria, which became engulfed in a nonphotosynthetic host cell. 26. Write about plant mitochondrial genome.(May-17) Plants possess mitochondrial genomes that are large and complex compared to animals. Nearly all animal mitochondrial genomes are about 16.5. kbp in length, whereas plant mitochondrial genomes range between 200-2,000 kbp. This is curious if we assume modern mitochondria originated from a common alpha-proteobacterial ancestor. Despite their size, plant mitochondrial genomes do not contain significantly more genes than their animal counterparts. Most of the additional DNA found in plant mitochondrial genomes consists of large introns, repeats and non-coding regions. Furthermore, plant mtDNA does not exist as large circular DNA molecules but mostly as a collection of linear DNA with combinations of smaller circular and branched molecules. Part -B 1. Write a detailed notes on the structural organization of chloroplast and mitochondria 2. Write notes on the structure of chloroplast and mitochondrial genome.(Nov/Dec2013) 3. Write notes on light reaction and explain it neatly. 4. What are C3 plants, explain the pathway neatly? 5. Write detailed notes on i). C4 and ii). CAM plants. 6. Explain, how a Glucose molecule is entering into the mitochondria? 7. Give a detailed account on TCA cycle and its energy balance. 8. What is cytoplasmic male sterility? Give a suitable example, the manifestation location and application. (Nov. 2011). 9. Explain in detail the cytoplasmic male sterility and how it will be useful in breeding hybrids. (May. 2014) 10. What is the molecular factor responsible for the Texas Male Sterile cytoplasm? Explain how (TMS) this is used in hybrid seed production. (Nov. 2014) 11. Write the gene content of chloroplast and explain how and what genes can be manipulated for higher photosynthetic efficiency. (Nov. 2011). 12. Give the general organization of chloroplast genome with a neat diagram, List and explain the protein complexes responsible for photosynthesis. (Nov. 2014) 13. Explain structure, function and genetic material of chloroplast. (May 2012)(May-17) 14. Write in detail about the Rubisco enzyme? (May 2012). 15. Describe in detail the assembly, mechanism and regulation of rubisco synthesis. (May. 2014) 16. What are the various protein complexes present in mitochondria, their role, gene location and coordination to carry out respiration? (Nov. 2012). 17. What is the unique feature of plant chloroplast genome organization give an example of Nod gene products and their role? (Nov. 2012). 18. Describe plastid genomes. Write a note on their evolution and utilization in plant genetic manipulation? (May. 2015) 19. Why is engineering of chloroplast and mitochondria more advantageous than compared to nuclear genes? Justify. Describe the different techniques that enable plastid engineering? (May. 2015) 20. Write a detailed essay on co- ordination, regulation & transport of protein. (May - 16) 21. Explain the structure and function of mitochondria in detail. (May - 16) 22. Explain in detail about plant genome regulation and its mechanism? (May - 17)

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 Unit III NITROGEN FIXATION

1. What are Diazotrophs? Diazotrophs are the microbes which are involved in the N2 fixation, these posses genes responsible for N2 fixation like nod, nif and hub genes ex. Rhizobium, Nostoc, etc. 2. List few anaerobic nitrogen fixer. Clostridium, Desulfovibrio, Klebsiella, etc. 3. Name few free living Diazotrophs Azospirillum amazonense, Azospirillum lipoferum, Beijerinkia, Derxia, etc. 4. List some of the organisms involved in biological nitrogen fixation. (May 2014, 2016, 2017) Azospirillum amazonense, Azospirillum lipoferum, Beijerinkia, Rhizobium, Nostoc, etc. 5. What is denitrification? This is a process in which ammonia, nitrate and nitrites are converted into free nitrogen eg. Thiobacillus denitrificans, Micrococcus denitrificans, etc. 6. List few organisms involved in converting nitrite to nitrates? Nitrobacter, Nitrocystis, Penicillium sps., etc 7. Name few organisms involved in converting Ammonia to nitrite. Nitrosomonas, nitrococcus,Nitrospira, etc. 8. What is ammonification? Dead and decaying plant and animals are acted upon by organisms like Bacillus ramosus, Bacillus vulgaris,Bacillus mesenterilus, etc. which converts various nitrogenous compounds into ammonia. 9. Write notes on Frankia. It is an actinomycetous organism, it form root nodules in non-leguminous plants like Alnus, Casurina, etc. The nodules formed are white in colour because of the lack of Leghaemoglobin. 10. Write notes on bacteroid formation. Inside the inner cortical region, rhizobium cells are enclosed in a membrane derived from the host cell and multiplied into 4- cells, finally become enlarged and pleuromorphic in nature called as Bacteroid. This may be swollen, star, irregular, club or y-shaped structures and is responsible for N2 fixation. 11. What are bacteroids? Give their significance. (Nov. 2011, May 2012, Nov. 2014) Inside the inner cortical region, rhizobium cells are enclosed in a membrane derived from the host cell and multiplied into 4- cells, finally become enlarged and pleuromorphic in nature called as Bacteroid. It avoids the accumulation of O2 there by assist the N2 fixation. 12. What are heterocysts? These are structures found in Heterocystous cyanobacteria and are specialised cells, which lack O2 evolving photosystem II, Ribulose biphosphate carboxylase and fewer amounts of photosynthetic pigments.Cell wall contain O2 binding proteins, since O2 inhibit N2 fixation this structure play a major role in the cyanobacteria ex. Nostoc, Anabaena, etc. 13. Write notes on nod genes. This gene is responsible for nodulation in leguminous plants, this gene is present in the plasmids of Rhizobium, it is closely associated with nif genes ex. nod A,nod B, nod C and nod D. 14. Write notes on Nitrogenase enzyme. It is involved in the conversion of N2 into ammonia, 3 types of Nitrogenase enzymes are available Among this Mo- nitrogenase posses Molybdo protein and ferro protein, Molybdo protein contain 2 Mo, 32 iron, 30 sulphur but Fe protein contain 4 iron, and 4 sulphur atoms. 15. What is the role of Hydrogenase enzyme in N2 fixation? Diazotrophs evolve hydrogen at the time of N2 fixation , this H2 is having inhibitory effect on N2 fixation, this H2 will be removed by the action of hydrogenase enzyme. 16. Name any two heterocystous cyanobacteria. Nostoc, Anabaena, etc. 17. What is LHB ? (Nov 2012, Nov. 2014) It is a red pigment just like the human haemoglobin in leguminous plants, red colour is due to the presence of iron , it act as a carrier of O2 and regulate O2 concentration and favours N2 fixation.

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 18. Differentiate white nodule and red nodule. In leguminous plants, red nodules are formed by Rhizopbium it is red colour due to the presence of iron, it act as a carrier of O2. But in non leguminous plants white nodules are formed by Frankia it is colourless because of the absence of iron. 19. Name few important factors which control nodulation. Concentration of inorganic nutrients, CO2 concentration , presence of ammonia, etc. 20. Write notes on Rhizobium. These are free living, gram negative, non sporulating, aerobic , motile, rod shaped bacterium. It is responsible for nodulation in legumes ex. Rhizobium japonium, R.leguminosarum.

21. Write notes on δ N It is a specialised sigma factor responsible for the recognition of promoter region in nif genes , nif genes contain 20 different genes organized into 7 operons , after recognizing the promoter it will be relieved from the core enzyme. 22. Differentiate sym plasmid and nod base. Rhizobial plasmid encoding nif genes and other genes , which is responsible for symbiosis is called sym plasmid. Each nod gene is proceded by a conserved DNA sequence called nod base. 23. Differentiate DRAT and DRAG. DRAT- Dinitrogenase reductase ADP- ribosyl transferase which catalyse ribosylation of ADP. DRAG - Dinitrogenase reductase activating glycohydrolase which catalyse the removal of ADP ribose from FE-protein. 24. Write notes on nod genes. Explain how nodulation takes place in leguminous plants. (May. 2014) Nodulation in leguminous plant is controlled by genes in the plasmids of Rhizobium, it is closely associated with nif genes , 4 nod genes like nod A,B,C & D among this nod A,B &C are called common nod genes. 25. Write notes on the important features of heterocysts. Heterocysts lack O2 evolving photosystem II, Ribulase biphosphate carboxylase and photosynthetic pigments, the cell wall posses O2 binding proteins. 26. How does a nodule structure aid in nitrogen fixation? (Nov. 2011). Nodule contain LHb which prevent the accumulation of O2 in the nodule, thereby it enhance N2 fixation. 27. Define HUP genes? (May 2012).(Nov/Dec2013) Diazotrophs evolve hydrogen at the time of N2 fixation , this H2 is having inhibitory effect on N2 fixation, this H2 will be removed by the action of hydrogenase enzyme. The gene responsible for the production of hydrogenase enzyme is called as HUP gene. 28. Name a plant species having stem nodules, and state its functional significance? (Nov 2012). Sesbania rostrata stem nodule increased their O2 consumption and nitrogenase activity at increasing concentrations of free dissolved O2 up to 15 nM. At these very low concentrations, sesbania stem nodule leghaemoglobin was better than soybean leghaerno- globin in the facilitation of O2 flux. 29. How do bacteroid help in nitrogen fixation? (May. 2015) The nitrogen-fixing nodule hosts symbiotic Rhizobium bacteroids, which function as specialized nitrogen fixing organelles that exchange fixed nitrogen for photosynthates. A physiological paradox arises from the aerobic requirements of bacteroid metabolism compared with the oxygen sensitivity of nitrogenase in the absence of a dedicated bacterial protective system. Protection against oxygen is provided by the nodule environment through a cortical diffusion barrier so that the main route of oxygen diffusion is through the nodule apex, which generates a longitudinal oxygen gradient (see the figure of a starch-stained alfalfa nodule; scale bar represents 200 m). As a result, the free oxygen concentration drops to less than 50 nM in the central nitrogen-fixing zone containing Rhizobium bacteroids. Oxygen diffusion is facilitated in the central zone by a high concentration of leghaemoglobin, and bacteroid respiration is made possible by the induction of a high affinity cbb3 oxidase. Therefore, Rhizobium bacteroids fix nitrogen in a microaerobic, nitrogen-rich environment,

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 which explains why oxygen, not nitrogen, is the main physiological regulatory factor for nif gene induction during symbiosis. 30. What is meant by stem nodulation? Give examples. May 2016 Rhizobia can establish a nitrogen-fixing symbiosis with plants of the Leguminosae family. They elicit on their host plant the formation of new organs, called nodules, which develop on the roots. A few aquatic legumes, however, can form nodules on their stem at dormant root primordia. The stem- nodulating legumes described so far are all members of the genera Aeschynomene, Sesbania, Neptunia, and Discolobium. Their rhizobial symbionts belong to four genera already described: Rhizobium, Bradyrhizobium, Sinorhizobium, and Azorhizobium. 31. What you understand by hybridoma technology?(May-17) Hybridoma technology is a method for producing large numbers of identical antibodies (also called monoclonal antibodies). ... In contrast to polyclonal antibodies, which are mixtures of many different antibody molecules, the monoclonal antibodies produced by each hybridoma line are all chemically identical. Part- B 1. What is nitrogen cycle, explain it clearly? 2. Write notes on the biological nitrogen fixation in detail. 3. Give a detailed account on the role of Rhizobium in nitrogen fixation. 4. Write notes on the molecular mechanism of nitrogen fixation. 5. Illustrate with suitable diagrams biological nitrogen fixation in detail. (May. 2014) 6. Write a detailed notes on DRAT and DRAG mechanism. 7. Give a detailed account on the regulation of nif genes with relevant example.(May-17) 8. What do you meant by gene regulation , explain the regulation of nitrogen fixation related genes. 9. Write notes on i). nod genes ii). nif genes iii). Hup genes iv). Bacteroid(Nov/Dec2013) 10. What is biofertilizer, explain it with neat example? 11. What are the various biotechnological approach for enhancing nitrogen fixation? 12. Give examples of NOD genes and their role, how nodulation genes are isolated by complementation? Briefly write on plant specificity of rhizobium with example. (Nov. 2011). 13. Explain by citing examples the genetic regulation of nitrogen fixation in the root nodules of leguminous plants. (May. 2014) 14. Explain the process of N2 fixation and genetic engineering of nitrogenous gene cluster.(Nov. 2011). 15. Explain how Nitrogenase gene cluster is engineered using gene complementation? (Nov. 2014) 16. Explain the process and biochemical mechanism involved in nitrogen fixation comment on ‘nod’ genes (May 2012). 17. What are the diazotrophs? How does it improve the soil fertility through nitrogen fixation? (May 2012). 18. How are nodulation genes isolated by gene complementation? Give examples of Nod gene products and their role. (Nov 2012). 19. Elaborate on the genetic engineering of nodulation genes. (Nov. 2014) 20. Give a list of Nif genes, their function and explain the process of nitrogen fixation (Nov 2012, May - 16). 21. Describe the function of Nitrogenase enzyme in biological nitrogen fixation?. Discuss the essentiality of enhancing its activity to improve nitrogen fixation? (May. 2015) (May - 16) 22. Explain in detail the process of biological nitrogen fixation and the chemistry involved with it. How can biotechnology help in enhancing the process? (May. 2015) 23. Discuss in detail about the different types of vectors used in plant gene transfer?(May-17)

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 Unit IV AGROBACTERIUM & VIRAL VECTORS

1. Write notes on Agrobacterium? It is a soil borne, gram negative bacterium it attacks many dicotyledonous plants and cause crown gall and hairy root disease . The disease causing ability resides in Ti & Ri plasmids, it is a commonly used vector for transgenic plant production. 2. What is T-DNA? (May 2016) Tranfer DNA (T-DNA) is a part of Ti plasmid at the time of transfer this region enter into host & integrate into the host genome. This region is responsible for hormone and plant metabolite synthesis. 3. List few important properties of Ti Plasmid. (May 2012). Ti plasmid posses important regions like T-DNA, vir-region, origin of ication, makes and restriction sites for different enzymes, etc. among this T-DNA posses the oncogenic region which codes for & biosynthesis and the vir genes are responsible for infection and the entry of pathogens into the host. 4. What is Agro infection? Infection of host plant by virus along with T.DNA of agrobacterium is called agroinfection. After infection viral genome replication occurs ex. Maize streake virus. 5. Explain the way in which T-DNA enters into plant cell? Release of phenolic compounds from the wounded plant tissue act as a signal and the cells will be acting as a competent cells. Virulent cells produce B-1,2 glycan and succinoglycans. Vir A product sense phenolic compounds as a result vir G is activated this will induce other vir gens. Vir D2 product act as an endonuclease & cut T-DNA , Vir D2, T-DNA along with VirB & Vir D4 product form a complex, this will be transfed into the plant cell through nuclear pore complex. 6. Write notes on advantages of A rizogens infection.  Better anchorage of plants.  Resist anoxia from flooding of soil  Increased drought resistance due to high root density .  Greater chance of interaction with siol borne fungi. 7. Write notes on binary vectors Cointegrate vectors are larger in size, hence the vir genes from the Ti plasmid and T-DNA containing The are separately inserted into plasmids. The above plasmids can be inserted into the Agrobacterium by triparental matings. 8. Explain palindromes. At the time of restriction enzyme digestion, short projections of sticky ends which are complementary to each other is produced these complementary strands are called as palindromes. 9. What is Alkaline phosphatase? During the gene transfer work, the restricted plasmid instead of joining with the foreign DNA it joins with the sticky end of the same plasmid this error can be avoided by treating it with alkaline phosphatase. 10. Write notes on PBR 322. It is a hybrid vector of pBR318 and 320. It contains origin of replication, resistant genes for ampicilin, tetracyclin, restriction sites for 20 different enzymes among this BamHI site occurs in tetracyclin resistance. 11. Different phage & cosmid vector? Bacteriphage DNA with its cos-site is used as a vector called phage vector, cosmids are hybrid vectors prepared by combining plasmid and phage DNA. Cosmid posses the cos site from phage. 12. Explain YAC? Yeast Artificial Chromosome, it is artificially constructed by adding Yeast episomal plasmid, Yeast integrating plasmid, yeast replicating plasmid, Yeast centromere and telomere in a single plasmid. More than 100 kb size of DNA can be cloned by using YAC. 13. Explain YAC and its use. It is Yeast artificial chromosome and artificially constructed by combining yeast episomal VTHT Page 14

BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 plasmid, yeast integrating plasmid, yeast replicating plasmid, yeast centromere and telomeric plasmid in a plasmid . More than 100 kb DNA can be cloned by using YAC. 14. Differentiate EcoRI and Bam HI. Both are restriction endonucleases but the site of action may vary, EcoRI specifically cuts at GAATTC CTTAA G BamHI specifically cuts at GGATCC CCTAGG 15. Write notes on Hind III enzymes It is a restriction endonuclease enzyme , it is isolated from Haemophilus influenza, it specifically cuts the DNA strands at AAGGTT TTCCAA 16. What is cointegrativre vector? Disarmed Ti plasmids can not be used as a vehicle because of larger size these plasmids are cloned into E.coli cells and gives an intermediate vector, this lack replication and conjugation, hence it is mixed with a helper plasmid using E.coli as aresult new vector called cointegrate is formed ex.pGV3850. 17. Write notes on plant virus. Virus which infect plants discovered in 1892 and found that mosaic virus can be tranmitted through the sap of an infected plant. In the year 1935 Stanley first crystallised plant virus. Most of the plant virus posses RNA as a genome. 18. Differentiate persistant and non pestistant virus? Non persistant virus spreads by mechanical means without any biological relationship between virus and host. Where as persistant viruses are transferred by vectors and multiply with in the vectors.example: Potato leaf roll Virus. 19. What is a virion? Complete assembly of an infections viral particle is called as virions. Each virus contain nucleic acid as a genome and it is well protected by a protein coat. Example: TMV 20. Write notes on BSMV. It is a rigid helicalal plant virus, 130nm long, 30nm wide . It infect Barley and cause this Barley strike mosaic virus disease . Protein coat posses 3 particles with different size 111nm, 128nm , 148nm, 60 sub units will be there in five turns of the helix and the genome is divided into 2-3 RNA fragments. 21. Write notes on CaMV. Cauliflower mosaic virus is a type member of Caulimovirus, it contain dsDNA as genetic material and is divided, it infects wide range of dicots. The genome contain 8 tightly packed genes among this 2 region are non essential this region can be replaced by the gene of interest,it is used as a viral vector for gene transfer 22. What are the salient features of viral vectors ?  It easily introduce the genomic material into the intact plant cell or tissue by simple rubbing.  Infected cells express high level of the recombinant DNA.  Viral infection is systemic, hence it allow the transgene expression through out the plant. 23. What are satellite vectors? Virus whch require the help of other activator or helper viruses for multiplication is called as satellite viruses ex. Satellite tobacco necrosis virus, these viruses can be used as a vector called as a satellite vectors. 24. Name any two suitable viral vectors. Geminivirus - Maize streak virus Caulimovirus - Cauliflower Mosaic virus 25. What is cross protection? Plants infected with one virus will be protected against infection by related virus. Viruses with mild symptoms are allowed to infect plants, which generate resistance to infection by more virulent virus. ex. Papaya ringspot virus and Zuchini yellow mosaic virus. 26. What is an IS element? VTHT Page 15

BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 In prokaryotes and some eukaryotes like Maize, specific DNA sequence move from one part to another, these mobile sequences are called as transposable elements or transposons, these are otherwise called as Insertion elements (IS). 27. Why are plant viral vectors not suited for transformation? (Nov. 2011, Nov 2014) Virus-induced gene silencing and (VIGS) and co-suppression are the important reasons. Viral vectors may also cause disease in some occasions. Whereas in Agrobacterial infection infection is easy and it never cause the disease. 28. How is Agrobacterium attracted to root and crown gall formed? (Nov. 2011, Nov. 2014) Release of phenolic compounds from the wounded plant tissue act as a signal and the cells will be acting as a competent cells. In order to cause gall formation, the T-DNA encodes genes for the production of auxin or indole-3-acetic acid via the IAM pathway. Genes for the production of are also expressed which stimulates cell proliferation and gall formation. 29. Define Rhizogenes? (May 2012). Agrobacterium Rhizogenes cause hairy root disease in plant. The Ri plasmids are isolated from this organism only and used for plant gene transformation. 30. What are octopine and nopaline plasmids? (May 2012). Agrobacterium tumefaciens contain the plasmid for the metabolism of octopine and nopaline are called as octopine and nopaline plasmids. 31. What are the limitations of plant viral vectors? (Nov. 2012). Virus-induced gene silencing and (VIGS) and co-suppression are the important reasons. 32. What are the plant pathogen recognition signals involved in the crown gall disease? (Nov 2012). Disease resistance depends on the ability of the plant to recognize a pathogen early in disease causing mechanism. Molecules that indicate the presence of the pathogen (elicitors) activate host receptors and several transduction pathways that relay the initial recognition signal through a series of cytosolic mechanisms. Acetosyringone is the best example. 35. Write about the importance of border sequences in causing tumorigenesis. (May 2014). The T-DNA region of the Ti Plasmid is marked by the presence of right and left border elements, these are direct repeats. Right border sequence when eliminated the virulence is affected, hence it is concluded that the virulence is governed by the righ border element. 36. What are viroids and what is its significance. (May 2014). Viroids are the smallest infectious pathogens known, consisting solely of short strands of circular, single-stranded RNA without protein coats. They are mostly plant pathogens, some of which are of economical importance. Viroid genomes are extremely small in size, ranging from 246 to 467 nucleobases. It easily infect plants. 37. Why is neomycin phosphotransferase a sought after system in plant genetic engineering? (May. 2015) Two neomycin phosphotransferase genes are used in selection of transformed organisms: the neomycin phosphotransferase I (nptI) gene and the neomycin phosphotransferase II (nptII) gene. The second one is the more widely used. It was initially isolated from the transposon Tn5 that was present in the bacterium strain Escherichia coli K12. The gene codes for the aminoglycoside 3'- phosphotransferase (denoted aph(3')-II or NPTII) enzyme, which inactivates by phosphorylation a range of aminoglycoside antibiotics such as:  kanamycin  neomycin  geneticin (G418)  paromomycin. NPTII is probably the most widely used selectable marker for plant transformation. It is also used in gene expression and regulation studies in different organisms in part because N-terminal fusions can be constructed that retain enzymatic activity. In animal cells, G418 and neomycin are used as selectable agents.[add a comment]

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 NPTII protein activity can be detected by enzymatic assay. In other detection methods, the modified substrates -the phosphorylated antibiotics- are detected by thin-layer chromatography, dot-blot analysis or polyacrylamide gel electrophoresis. 38. State the advantages and disadvantages of Agrobacterium mediated plant transformation? (May. 2015) Advantages:  Natural means of transfer  Capable of infecting intact plant organs  Capable of transferring large fragment of DNA  Stability of gene transferred is excellent Disadvantages:  Limited host range  Imp food crops cannot be infected  Cells in the tissue that are able to regenerate cannot be transformed 33. State the significance of satellite sequence in cross protection? (May - 16) Some viruses have specific feature to contain, in addition to their genomic RNA, a small RNA molecule known as satellite RNA (S-RNA). The S-RNAs require the company of a specific 'helper' virus (closely related virus) for their replication. S-RNA does not have sequence to encode CP. They are encapsulated in the coat protein of their helper virus or satellite vi-ruses which encode their own coat protein. Due to ability to modify disease symptoms S-RNAs now have point of attention in genetic engineering. Most of the S-RNAs decrease the severity of viral infection, presumably through interference with viral replication. By this method, tomato, a number of pepper varieties, cucumber, eggplant, cabbage and tobacco plants against CMV have been protected. The first time S-RNA induced attenuation of viral symptoms involved the introduction of cDNA copies of CMV S-RNA into the genome of tobacco plants. Part- B 1. What are the various vital stages involved in rDNA technology? 2. Write notes on the role of Agrobacterium mediated gene transfer. 3. What are the molecular events that occur during Agrobacterium infection? 4. Write notes on i).Cointegerate vector and ii).Binary vector. 5. Explain i). Inplanta transformation ii).Co-culture iii).T-DNA iv). Vir genes. 6. Write notes on various physical, chemical and biological gene transfer techniques. 7. Write notes on i).Particle gun bombardment ii).CaMV iii). YAC. 8. What is the role of various marker genes in rDNA technology? 9. Write detailed notes on the various physical gene transfer techniques. 10. Give an account on the role of various viral based vectors. (May 17) 11. Describe the structure and properties of cauliflower mosaic virus and Gemini virus? (May 2012). 12. Describe how T – DNA is excised and transferred from agrobacterium and integrated into plant genome? (May 2012). Or 13. Elucidate the process of DNA transfer and integration by Agrobacterium with suitable diagrams.(Nov 2012).(Nov/Dec2013) 14. What are the modifications done on Ti plasmids for converting it to a transformation vector? Draw the map of a Agrobacterium binary vector (Nov 2012). 15. Describe in detail the Agrobacterium mediated genetic transformation in dicot plants. (May 2014) 16. Discuss the different types of viral vectors used in genetic engineering of plants and its applications. (May 2014, May 15) 17. Draw the structure of a binary vector and label the components and mention the function of each component. (Nov. 2014) 18. Elaborate the various method plant transformation methods and vectors available to engineer plants. Discuss their merits and demerits. (Nov. 2014, May - 16) 19. Justify Agrobacterium mediated plant transformation using both Binary vector and Co- Integrate vector (May. 2015, May - 16) 20. Write in detail about the Ti plasmid mediated gene transfer in plants.(May-17) VTHT Page 17

BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 Unit V APPLICATION OF PLANT BIOTECHNOLOGY Part – A

1. What is totipotency?(May 2014, May - 16) It is the capacity of a plant cell to develop into an organism by degenaration and regenaration. This property is utilized for plant tissue culture, fusion, artificial seed production, etc. 2. What is the role of various plant hormones in plant tissue culture? (Nov. 2012). These are otherwise called as Phytohormons, essential for the growth and development of plants. Different harmones like, Shoot inducing , root inducing and inducing harmones are available for plant tissue culture ex. Zeation, IAA, NAA, 24-D, etc. 3. Which hormones decide the fate of shoot and root growth? Discuss. (Nov. 2014) Auxin – root induction Cytokinis – shoot formation 4. Differentiate explants and callus. Any plant part or tissue used as seeding material is called as an explant. Explant after degeration gives undifferentiated mass of tissue called as callus. 5. What is somatic embryos? Embryo developed from somatic tissue through tissue culture is called somatic embryos.Two type of somatic embroyos formation direct in which explant is directly modified into embryo. In other case explants will be modified in to embryo through callus. 6. Explain ? Formation of oragans like shoot, roots and leaves from the explant or callus is called as organogenesis, phytohormones are used to induce different organs. 7. Differentiate cell and protoplast. Cell is the basic unit of any living organisms, Proto is a cell with out cell wall. Different enzymes are used to liberate protoplast from the cell. It is used for cell suspension culture, protoplast fusion, etc. 8. Different cybrid and hybrid. When two nuclei of two different cell fuse it gives hybrid. Fusion of cytoplasm without the fusion of nuclei leads to the formation of cybrids. 9. What is androgenesis? Formation of plants from pollen grain is called as androgenesis and the plants will be haploid in nature. 10. What is an artificial seed? (May 2012). Somatic embryos encapsulated by synthetic material like polyethylene, polyoxol, etc.,are called as artificial seeds.Artificial seeds are developed in Eukalyptus. This can be used as a seeding material but the is very low. 11. Write notes on lux genes. It is a luciferase gene , in bacteria two lux genes - lux A and lux B , but in fire fly only one lux gene is present, which catalyse the oxidation of luciferin as a result bioluminescense occur. 12. Write notes on Lipofection? Introduction of target DNA in to the host through Liposomes are called lipofection.Liposome enter through endocytosis and plasmid DNA integrate into the host genome.This will protect in DNA against nucleases. ex.: successful in Tobacco, Petunia, etc. 13. Write notes on microprojetile or particle gun bombardent. DNA is inserted into target cell or tissue (along with microprojectiles). Microprojectiles are the tungston or gold particle with 1-3 um dia. 2um size coated with DNA, it is acclerated into plant cells at high velocity by pressurised helium gas. 14. What are macroprojectiles? Plastic membrane having 2.5 cm dia, 0.06mm memberane during gun bambardmant macroprojectiles with DNA will be transferred onto the macrocarriers from this it will be acclerated into a plant tissue. 15. Write notes on antimicrobial proteins. Proteins with antibacterial and antifungal activities are called as antimicrobial proteins ex.Lysozyme, thionin, defensins,etc.

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 16. Name few transgenic methods adopted to avoid viral diseases. Expression of coat protein gene inside the host, Antisense RNA, Ribozymes, gene silencing. 17. Explain antisense RNA and its role. These are complementary sequences to the mRNA, this will hybridise with the mRNA and inhibit translation and other related process, hence it is used to arrest the multiplication of pathogens. 18. What is RNA interference? In post translational gene silencing the dsRNA sliced by RNA endonuclease resulting in the production of small sense (21-25 bp) and antisense RNA called SiRNA, this can be transported through out the plant and guid the different ribonuclease to cut & degrade homologous region called as RNA interference. 19. What are defensins? Defensins are antimicrobial proteins synthesized in plants to prevent pathogens ex. AtNH the time of seed germination defensins are released from the seeds inorder to [protect the seeds from fungal attack. 20. What are phytoalexins? These are antipathogenic chemicals, synthesized on infection which inhibit the invading pathogens. ex. Methylated flavanone 21. Name few pathogen related proteins. Chitinase, Ribosomal inhibiting proteins, B-1,3-glucanases and defensins. 22. What do you mean by systemic acquired response? Infection by a pathogen induces local defence pathway leads to the induction of cellular signals which causes systemic response termed as systemic acquired response ex. Salicylic acid released as signal will reach different parts through phloem where it induce the expression of SAR genes 23. What is “”?(Nov/Dec2013) It is a transgenic tomato available U.S market. In which polygalacturanase gene which is responsible for fruit softening is inactivated by antisense RNA, it delays fruit ripening in tomatos. 24. Name a herbicide tolerant transgenic plant, the transgene and its function? (Nov. 2012). The herbicide glyposate and other herbisides inhibit yield of the major crop plants, in the case of glyposate it inhibit EPSP enzyme, the transgenic plants with more amount of this gene product will resist the effect of this herbicide ex.Transgenic tomatos,potatos,etc. 25. List some edible vaccines, which are under clinical trails. (May. 2014) Production of vaccines in edible plant parts and transformation of plants with small parts of the antigen may also be used as vaccines called as subunit vaccines. ex. Oral Cholara vaccine CT- B contain only B sub unit of the toxin. ex. Transgenic Banana,Tomato. 26. Mention a plant based recombinant product and reason for the plant of choice. (Nov. 2011). Hirudin – recombinant tobacco, Vitamin A- . 27. Mention the at least two oilseed crops modified for biofuel. (Nov. 2014) Modified Jatropha offers improved qualities for fuel, maize trademarked Enogen that has been genetically modified to convert its starch to sugar for ethanol and poplar trees genetically engineered to contain less lignin to ease conversion into ethanol. 28. What do you understand by “ Round up ready soy bean”?. (May. 2015) Two key elements needed for the development of commercially viable glyphosate-tolerant crops are: a resistant target enzyme sufficient expression of that enzyme within the transgenic plant”. When incorporated into the genome of RR-susceptible plants, the Roundup ReadyTM gene (CP4 EPSPS) confers resistance to glyphosate

29. What is gene silencing ? always will not express the gene products stably, in some cases it became unstable and mask the expression of other genes this process is called as gene silencing. In TGS it has holology

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 with the promoter region it lesds to methylation and alteration in the chromosome conformations. In PTGS gene silencing increasd with increase in transcription of the transgene here inhibition of both transgene and endogenous gene occur. 30. List out some therapeutic protein obtained from plants? (May - 16). Potential application or human protein Platn Host Protein Anticoagulant Tobacco Protein C Thrombin Inhibitor Tobacco Hirudin Neutropenia Tobacco Granulocyte- Macrophage- colony stimulating factor 30. Write notes on pesticide resistant transgenic plants. Transgenic plants with pest resistant genes, cry genes isolated from Bacillus thuringiensis was introduced into plants for arresting lepidopterna larval attack. ex. Bt.. 31. Write notes on Golden rice. Transgenic rice with Beta carotein content is called as Golden rice, it is yellow or brown in colour. In which gene for carotene is isolated from daffodil is inserted into rice through Agrobacterium based vector. 32. What are edible vaccines?(May 17) Production of vaccines in edible plant parts is called as edible vaccines, gene encoding an entire antigen is isolated and introduced into the plants through Agrobacterium as a vector, vaccines expressed in plants can be eaten as raw. ex. Transgenic tomato, banana, etc. 33. What are the categories of ? How do they influence organogenesis?(Nov.2011). There are five major classes of plant hormones – Abscisic acid, Auxin, Gibberellins, Cytokinins and Ethylene. Plant hormones are used to initiate callus growth. Specific auxin to cytokinin ratios in plant tissue culture medium give rise to an unorganized growing and dividing mass of callus cells. The hormones at a specific ratio also induce shoot and root growth. 35. Define the term callus.(May 17) Undifferentiated mass of cells developed using explant when its being introduced into a proper medium. Part- B 1. Write detailed notes on plant tissue culture and its importance.(May 17) 2. Write notes on embryo culture and somatic embryogenesis. 3. Write notes on the role of nutrients and growth hormones in plant tissue culture. 4. Write detailed notes on i). Artificial seeds ii). Terminator technology iii). Somaclonal variation 5. Write notes on the various recent approaches to control viral infection. 6. Write a detailed notes on the role of B.thuringiensis in the development of pest resistant plants. 7. Write detailed notes on role of genetically modified plants in food security. 8. Explain edible vaccines and clinically important proteins. 9. Write detailed notes on i). Golden rice ii) Flaur saur. iii). Salt resistant plants. 10. What is molecular farming? Describe the oleosin system for hirudin and insulin production. (Nov.11). 11. Give three categories of , their mode of action and strategy for herbicide tolerant plant development. (Nov. 2011, Dec2013, May - 16) 12. How can transgeneic be derived with non pathogen – derived resistance. (May 2012). 13. (i) Explain in detail about molecular pharming? (May - 16) (ii) Give an account on the importance of therapeutic products? (May 2012). 14. Describe the general structure of Bt toxin (cry protein) and list various cry proteins identified so far and exploited for pest resistance. (Nov 2012). 15. How are therapeutically important compounds produced in plants on a large scale? Discuss the merits and demerits of using plant system (Nov 2012). 16. What are the various method of plant Tissue culture available? Discuss the applications of each one of them. (Nov. 2014).

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 17. Explain how plants can be used as a for producing therapeutics simultaneously discuss the limitations also. (Nov. 2014). 18. Among the various tissue culture techniques “micro propagation produces. (May. 2014). 19. How will you produce a BT resistant plant? Explain the process by citing an example.(May. 2014) 20. Discuss plant genetic engineering as a revolutionary process to make ands meet in terms of challenges relating to nutrition, environment and therapeutics?. (May. 2015) 21. Justify – “plants as ”. (May. 2015) 22. Write a detailed account on the strategies used in the development of transgenic plants . Explain with an example.(May 17)

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 Unit-I Part-B

1. Write notes on the condensation of Eukaryotic DNA. ( Nov/Dec 2013) Elaborately discuss about the structural organization of nucleosome and its dynamics. (May. 2014) DNA is made of double helical structure two right handed helical polynucleotide chain run opposite direction and two strands are held together by hydrogen bonds. Nucleosomes are bead like structures, each bead contain around 200 bp DNA , RNA and histonic and non histonic proteins. Histonic proteins are responsible for DNA condensation.These are basic proteins, 10 – 20 of aminoacids are basic aa’s like Arginine and Lysine. H2a, H2b, H3,H4 are involved in octomer formation, Molecular nature of DNA, histones and formation of nucleosome

SOLENOID STRUCTURE Interphase chromosome is 30 nm thick ,it is formed due to the folding of nucleosome chain into a solenoid structure , each turn is having 6 nucleosomes and a central hole. Scaffold formation due to 3rd level of condensation

2. What do you mean by replication, explain it neatly? DNA replication is the process of copying a double-stranded DNA molecule to form two double- stranded molecules. It is the formation of DNA from DNA by the presence of a template and DNA polymerases. In linear mode of replication , in the lagging strand replication is discontinuous these fragments are called okazaki fragments It is the type of replication occurs in circular DNA, in which the intermediate resembles the Greek letter Ɵ. In this mode the newly formed strand rolls around the circular template, hence called as rolling circle replication

3. Write notes on the Eukaryotic translation and post translational modifications./ VTHT Page 22

BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 Give a detail account on the translation mechanisms of eukaryotic system. (May 2014) Translation is the protein synthesis,occurs in the presence of template m RNA, enzymes, protein factors, etc initiation of translation in the presence of eIF1,eIF2,etc. pre initiation complex formation Elongation of polypeptide chain, role of eEF1, eEF2, etc. When the peptide bond formation is over, the acylated tRNA from A-site moved into the P-site this process is called as translocation. Peptide bond formation between aminoacids in A and P site occur in the prescence of an enzyme peptidyl transferase, this reaction occur in the 50s ribosomal subunit hence it is called as peptidyl transferase center Post translational modifications - Removal of methionine, acetylation of N- terminal aminoacid, hydroxylation of proline and lysine, glycosylation ,etc.

4. What is transcription, explain Eukaryotic transcription in detail. Eukaryotic transcription is more complex than prokaryotic transcription. For instance, in eukaryotes the genetic material (DNA), and therefore transcription, is primarily localized to the nucleus, various concerved sequences like TATA, CAT are also playing major role. Name Location RNA transcribed RNA Polymerase I (Pol I, nucleolus Larger ribosomal RNA Pol A) (rRNA) (28S, 18S, 5.8S) RNA Polymerase II (Pol II, nucleus messenger RNA (mRNA) Pol B) and most small nuclear RNAs (snRNAs) RNA Polymerase III (Pol nucleus (and possibly the transfer RNA (tRNA) and III, Pol C) nucleolus-nucleoplasm other small RNAs interface) (including the small 5S rRNA)

5. Differentiate post transcriptional and tanslational modifications in detail. Explain the various post- transcriptional and translational modifications that regulate gene expression in detail. (Nov 2012).(Nov/Dec2013). Post transcriptional: Removal of introns in Yeast tRNA and addition of CCA sequence, etc. Addition of 5’ capping and 3’ poly A tailing and removal of introns in mRNA Removal of introns in rRNA and the role of ribozymes. Post translational: Removal of methionine, acetylation of N-terminal aminoacid, hydroxylation of proline and lysine, glycosylation ,etc.

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19

6. Explain neatly on i). Co-translational and ii). Post translational translocations. i). Co-translational: The N-terminal signal sequence of the protein is recognized by a signal recognition particle (SRP) while the protein is still being synthesized on the ribosome. The synthesis pauses while the ribosome-protein complex is transferred to an SRP receptor on the endoplasmic reticulum (ER), a membrane-enclosed organelle. There, the nascent protein is inserted into the Sec61 translocation complex (also known as the translocon) that passes through the ER membrane. The signal sequence is immediately cleaved from the polypeptide once it has been translocated into the ER by signal peptidase in secretory proteins. This signal sequence processing differs for some ER transmembrane proteins. Within the ER, the protein is first covered by a chaperone protein to protect it from the high concentration of other proteins in the ER, giving it time to fold correctly. Once folded, the protein is modified as needed (for example, by glycosylation), then transported to the Golgi apparatus for further processing and goes to its target organelles or is retained in the ER by various ER retention mechanisms. Addition of short aminoacid sequence at C-terminal end a tetrapeptide like KDEL is added, in yeast HDEL or DDEL is added. ii). Post translational translocations: Even though most proteins are cotranslationally translocated, some are translated in the cytosol and later transported to their destination. This occurs for proteins that go to a mitochondrion, a chloroplast, or a peroxisome (proteins that go to the latter have their signal sequence at the C terminus). Also, proteins targeted for the nucleus are translocated post-translation. They pass through the nuclear envelope via nuclear pores. Translocation of proteins in chloroplast, mitochondria, peroxisomes, etc. Importance of TIM , Tom proteins, etc.

7. Give a detailed account on transcription initiation, elongation and termination of a eukaryotic gene? (May 2012).( Nov/Dec 2013) Draw the structure of a eukaryotic gene and promoter structure and explain the stage of transcription. (Nov. 2014) Eukaryotic transcription is carried out in the nucleus of the cell by one of three RNA polymerases, depending on the RNA being transcribed, and proceeds in three sequential stages: 1. Initiation 2. Elongation 3. Termination.

8. Explain complex organization of genetic material in a plant cell with illustration. (May 2012)(May.2015). Eukaryotic Genome Complexity

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19

Refer portal for notes. SOLENOID STRUCTURE Interphase chromosome is 30 nm thick,it is formed due to the folding of nucleosome chain into a solenoid structure , each turn is having 6 nucleosomes and a central hole.Scaffold formation due to 3rd level of condensation 9. Explain the various post- transcriptional and translational modifications that regulate gene expression in detail. (Nov 2012).( Nov/Dec 2013) (May.2015) Post transcriptional: Removal of introns in Yeast tRNA and addition of CCA sequence, etc. Addition of 5’ capping and 3’ poly A tailing and removal of introns in mRNA Removal of introns in rRNA and the role of ribozymes. Post translational: Removal of methionine, acetylation of N-terminal aminoacid, hydroxylation of proline and lysine, glycosylation ,etc. 10. Elaborate the various levels of gene regulation in eukaryotic protein coding genes. (Nov. 2014) A) Overview of Eukaryotic Gene Expression 1) The Flow of Genetic Information (gene expression): In general, the information encoded in DNA is transcribed into RNA and translated into proteins. 2) The DNA of different cell types is generally similar in both amount and type (notable exceptions will be discussed). 3) Each eukaryotic cell expresses only a small percentage of the genes it contains. a) Some genes (the so-called “house-keeping genes”) are likely (constitutively) expressed in all cell types since certain proteins (and RNAs) are involved in the basic metabolic processes common to all cell types. b) Other genes are expressed in one cell type but not another (e.g. certain immune cells normally synthesize antibodies but neurons do not). c) Thus, different cell types arise because of differential gene expression and the RNA and protein content of different cell types shows considerable variation. 4) Gene Expression Patterns are Complex and Dynamic a) tissue specific (basis for different cell types) b) developmental (temporal and spatial expression patterns) c) environmental (induction in response to external stimuli) 5) Major Differences in Prokaryotic and Eukaryotic Gene Regulation a) presence of a nucleus in eukaryotic cells b) multiple copies for many eukaryotic genes; large amount of non-coding DNA. c) genes are interrupted by non-coding DNA, which is transcribed d) coupled (proks) vs. uncoupled (euks) transcription/translation reactions e) polycistronic (more than one protein/mRNA; common in proks) vs. monocistronic (one protein/mRNA) in euks f) extensive modification of eukaryotic mRNAs g) multicellular vs. unicellular environments/responses B) Control Of Gene Expression Is Exerted At Multiple Levels TWO MAIN DIVISONS OF CONTROL: 1) Changes in DNA Content or Position 2 a) The general rule isthat the DNA of different cell types does not vary in either amount or type b) However, highly specialized cases are known to exist where DNA loss, rearrangement, and amplification profoundly influence gene expression in isolated situations. 2) Changes in Gene Activity (expression patterns) a) The general method of gene regulation b) Regulation is known to occur at several different points of a multi-step gene expression pathway. VTHT Page 25

BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 Four main levels of control include: 1. Transcriptional control: Detemines if, how much, and when an mRNA is made. 2. Post-transcriptional control: Determines if, how much, and when an mRNA is available for translation into a protein. 3. Translational control: Determines if, how much, and when a protein is made. 4. Post-translational control: Determines if, how much, and when a protein is functional. C) Control at the Level of Changes in DNA Content or Position 1) Gene Loss: (In rare instances, cells dispense with unneeded DNA) a) Mammalian Erythrocytes: The best known case occurs in the development of the mammalian erythrocyte (red blood cell). During differentiation of the erythrocyte from an erythroblast, the cell destroys its nucleus and its entire DNA. The resulting cell continues to produce large amounts of globin (the protei component of oxygen-carrying hemoglobin) from the pre-synthesized mRNAs but cannot produce other proteins (erythrocytes live for only short periods of time in your body). The loss of DNA in erythrocytes causes a specialized cell that functions to carry oxygen through the blood to tissues.

b) Developmental Loss of specific chromosomes is known to occur in early development of certai nematodes, crustaceans and insects. In these cases, it is the somatic cells that lose certain chromosomes. The germ cells (which will give rise to subsequent generations) retain the entire genome. 2) Gene amplification: (used in rare instances when proteins are needed more rapidly than the chromosomal number would permit) a) Developmentally regulated amplification of rRNA genes Amplification or DNA is driven by the need for massive amounts of ribosomes during . In frog oocytes, there is selective amplification of genes encoding ribosomal RNAs. During development of the frog oocyte, the number of copies of DNA encoding ribosomal RNA increases from 900 to up to 2 million. The extrachromosomal copies of rDNA in each oocyte is present in ~1,500 extrachromosomal nucleoli which synthesize ~1012 ribosomes. b) c-myc proto-oncogene amplification Some genes such as the proto-oncogene c-myc (a transcription factor) have been observed to be amplified to many copies within a particular cell leading to overexpression of the regulatory protein and tumor (cancer) formation. The mechanism by which extra copies of the myc genes are generated is unclear bu appears to be the result of over-replication and subsequent amplification bynonhomologous exchange. c) Selective amplification of eggshell (chorion) genes in fruit flies. Programmed activation of DNA replication specifically at the chorion genes leads to a 64-fold amplification of the genes to enable huge amounts of the proteins to be synthesized

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 Unit II Part-B 1. Write a detailed notes on the structural organization of chloroplast and Mitochondria. (Nov/Dec 2013)

Chloroplasts are organelles found in plant cells and eukaryotic algae that conduct photosynthesis. Chloroplasts absorb light and use it in conjunction with water and carbon dioxide to produce sugars, the raw material for energy and biomass production in all green plants and the animals that depend on them, directly or indirectly, for food. Chloroplasts capture light energy to conserve free energy in the form of ATP and reduce NADP to NADPH through a complex set of processes called photosynthesis. Chloroplast genome is a double stranded, circular DNA. Mitochondrial structural organization

Mitochondrion (plural mitochondria) is a membrane-enclosed organelle found in most eukaryotic cells. These organelles range from 0.5 to 1.0 micrometer (μm) in diameter. Mitochondria are sometimes described as "cellular power plants" because they generate most of the cell's supply of adenosine triphosphate (ATP), used as a source of chemical energy. In addition to supplying cellular energy, mitochondria are involved in a range of other processes, such as signaling, , cell death, as well as the control of the cell cycle and cell growth.

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2.What is cytoplasmic male sterility? Give a suitable example, the manifestation location and application. (Nov. 2011). Explain in detail the cytoplasmic male sterility and how it will be useful in breeding hybrids. (May. 2014). Cytoplasmic male sterility (CMS), a condition under which a plant is unable to produce functional pollen, is widespread among higher plants. CMS systems represent a valuable tool in the production of hybrid seed in self- pollinating crop species, including maize, rice, cotton, and a number of vegetable crops. Hybrids often exhibit heterosis, more commonly known as hybrid vigor, whereby hybrid progeny exhibit superior growth characteristics relative to either of the parental lines. CMS systems can be of considerable value in facilitating efficient hybrid seed production. CMS is a maternally inherited trait that is often associated with unusual open reading frames (ORFs) found in mitochondrial genomes In many cases, it has been found that male fertility can be restored by nuclear-encoded fertility restorer (Rf) gene(s).  Barnase/barstar system for engineered male sterility –(explain) 3. Write the gene content of chloroplast and explain how and what genes can be manipulated for higher photosynthetic efficiency. (Nov. 2011).  Gene content and protein synthesis  Chloroplast genome reduction and gene transfer  Protein synthesis

4. Explain structure, function and genetic material of chloroplast. (May 2012). Chloroplasts have their own DNA,often abbreviated as ctDNA, or cpDNA.It is also known as the plastome. Its existence was first proved in 1962,and first sequenced in 1986—when two Japanese research teams sequenced the chloroplast DNA of liverwort and tobacco.Since then, hundreds of chloroplast DNAs from various species have been sequenced, but they're mostly those of land plants and green algae— glaucophytes, red algae, and other algal groups are extremely underrepresented, potentially introducing some bias in views of "typical" chloroplast DNA structure and content.  Molecular structure  Inverted repeats  Nucleoids  Photosynthesis

5. Write in detail about the Rubisco enzyme? (May 2012).  Wealth of Rubisco Structures and Sequences VTHT Page 28

BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 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. 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. Interactions in larger subunit  Mutational Approaches 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 Ω. 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 N-terminal domain and at the interface between large and small subunits. 6. What are the various protein complexes present in mitochondria, their role, gene location and coordination to carry out respiration? (Nov. 2012). The Energy obtained through the transfer of electrons (blue arrows) down the ETC is used to pump protons (red arrows) from the mitochondrial matrix into the intermembrane space, creating an electrochemical proton gradient across the mitochondrial inner membrane (IMM) called ΔΨ. This electrochemical proton gradient allows ATP synthase (ATP-ase) to use the flow of H+ through the enzyme back into the matrix to generate ATP from adenosine diphosphate (ADP) and inorganic phosphate. Complex I (NADH coenzyme Q reductase; labeled I) accepts electrons from the Krebs cycle electron carrier nicotinamide adenine dinucleotide (NADH), and passes them to coenzyme Q (ubiquinone; labeled Q), which also receives electrons from complex II (succinate dehydrogenase; labeled II). UQ passes electrons to complex III (cytochrome bc1complex; labeled III), which passes them to cytochrome c (cyt c). Cyt c passes electrons to Complex IV (cytochrome c oxidase; labeled IV), which uses the electrons and hydrogen ions to reduce molecular oxygen to water. Four membrane-bound complexes have been identified in mitochondria. Each is an extremely complex transmembrane structure that is embedded in the inner membrane. Three of them are proton pumps. The structures are electrically connected by lipid-soluble electron carriers and water-soluble electron carriers. The overall electron transport chain:

NADH → Complex I → Q → Complex III → cytochrome c → Complex IV → O2 ↑ Complex II ↑ FADH2

 Complex I  Complex II  Complex III

QH2 + 2 cytochrome c (FeIII) + 2 H+in → Q + 2 cytochrome c (FeII) + 4 H+out

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19  Complex IV

Coupling with oxidative phosphorylation

Depiction of ATP synthase, the site of oxidative phosphorylation to generate ATP.

7. What is the unique feature of plant chloroplast genome organization give an example of Nod gene products and their role? (Nov. 2012). Chloroplast Genome Organization All angiosperms and land plants have cpDNAs which range in size from 120-160 kb; three expceptions are: Species Size (kb) N. accuminati 171 Duckweed 180 Geranium 217 Multimer Relative Abundance Percent Monomer 1 67.5 Dimer 1/3 22.5 Trimer 1/9 7.5 Tetramer 1/27 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  Evolutionary Changes of cpDNA VTHT Page 30

BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 Nod gene: A precise exchange of molecular signals between the host plant and rhizobia over space and time is essential for the development of effective root nodules. The first apparent exchange of signals is involved in the secretion of phenolic compounds, flavonoid and/or isoflavonoid, by host plants (Peters and Verma, 1990). These specific plant exudates activate the nod gene expression of rhizobia mediated by the nodD regulatory gene product (Peters et al., 1986). The capacity of a flavonoid to interact with a nodD gene product is strongly affected by its molecular structure. Specific flavonoid molecules such as naringenin and hesperetin are normally present in the rhizosphere of pea and lentil, and induce nod gene expression of R. leguminosarum. As a result of nod gene induction, a lipochitin oligosaccharide (Nod factor) is produced by the bacterial symbiont which, in turn, elicits root hair deformation and cortical cell division in the plant root, the early steps in nodule formation (Sanjuan et al., 1992).

14. Describe in detail the assembly, mechanism and regulation of rubisco synthesis. (May. 2014)

Ribulose-1,5-bisphosphate carboxylase/oxygenase, commonly known by the abbreviation RuBisCO, is an enzyme involved in the first major step of carbon fixation, a process by which atmospheric carbon dioxide is converted by plants to energy-richmolecules such as glucose. In chemical terms, it catalyzes the carboxylation of ribulose-1,5-bisphosphate (also known as RuBP). It is probably the most abundant protein on Earth. RuBisCO is the most abundant protein in leaves, accounting for 50% of soluble leaf protein in C3 plants (20–30% of total leaf nitrogen) and 30% of soluble leaf protein in C4 plants. The enzyme usually consists of two types of protein subunit, called the large chain (L, about 55,000 Da) and the small chain (S, about 13,000 Da). The large-chain gene (rbcL) is part of thechloroplast DNA molecule in plants.[4] There are typically several related small-chain genes in the nucleus of plant cells, and the small chains are imported to the stromal compartment of chloroplasts from the cytosol by crossing the outer chloroplast membrane.[5][6] The enzymatically active substrate (ribulose 1,5-bisphosphate) binding sites are located in the large chains that form dimers as shown in Figure 1 (above, right) in which amino acids from each large chain contribute to the binding sites. A total of eight large-chains (= 4 dimers) and eight small chains assemble into a larger complex of about 540,000 Da.[7] In some proteobacteria and dinoflagellates, enzymes consisting of only large subunits have been found. Magnesium ions (Mg2+) are needed for enzymatic activity. Correct positioning of Mg2+ in the active site of the enzyme involves addition of an "activating" carbon dioxide molecule (CO2) to a lysine in the active site (forming a carbamate).[9] Formation of the carbamate is favored by analkaline pH. The pH and the concentration of magnesium ions in the fluid compartment (in plants, the stroma of the chloroplast[10]) increases in the light. The role of changing pH and magnesium ion levels in the regulation of RuBisCO enzyme activity is discussed below. During carbon fixation, the substrate molecules for RuBisCO are ribulose-1,5-bisphosphate, carbon dioxide (distinct from the "activating" carbon dioxide). When carbon dioxide is the substrate, the product of the carboxylase reaction is a highly unstable six-carbon phosphorylated intermediate known as 3-keto-2- carboxyarabinitol-1,5-bisphosphate, which decays virtually instantaneously into two molecules of glycerate-3- phosphate. Rate of enzymatic activity RuBisCO is slow, being able to fix only 3-10 carbon dioxide molecules each second per molecule of enzyme. The reaction catalyzed by RuBisCO is, thus, the primary rate-limiting factor of the Calvin cycle during the day. Nevertheless, under most conditions, and when light is not otherwise limiting photosynthesis, the speed of RuBisCO responds positively to increasing carbon dioxide concentration. However, our descriptive knowledge will become more usable when we can translate them into quantitative models that can enable us to calculate the outcome of the reaction under a given condition. Since RubisCO reacts with RuBP (ribulose 1,5 bisphosphate) first to produces enediol and next with CO2 that after some intermediate changes produces PGA (3- phosphoglycerate), a biochemical model is developed [13] to represent the effects of these steps quantitatively. VTHT Page 31

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Since carboxylation or fixation of CO2 is possible only after the synthesis of enediol, thus it is suggested that the role of RubisCO is to produce enediol that is carboxylase and oxygenase (EnCO). Accordingly, RubisCO is called enolase-phosphglycerase (EPGase) since it is neither carboxylase nor oxygenase. Regulation of its enzymatic activity: RuBisCO is usually only active during the day as ribulose 1,5-bisphosphate is not regenerated in the dark. This is due to the regulation of several other enzymes in the Calvin cycle. Regulation by ions[edit] Upon illumination of the chloroplasts, the pH of the stroma rises from 7.0 to 8.0 because of the proton (hydrogen ion, H+) gradient created across the thylakoid membrane.[14] At the same time, magnesium ions (Mg2+) move out of the thylakoids, increasing the concentration of magnesium in the stroma of the chloroplasts. RuBisCO has a high optimal pH (can be >9.0, depending on the magnesium ion concentration) and, thus, becomes "activated" by the addition of carbon dioxide and magnesium to the active sites as described above. Regulation by RuBisCO activase In plants and some algae, another enzyme, RuBisCO activase, is required to allow the rapid formation of the critical carbamate in the active site of RuBisCO. Regulation by carbon dioxide Since carbon dioxide and oxygen compete at the active site of RuBisCO, carbon fixation by RuBisCO can be enhanced by increasing the carbon dioxide level in the compartment containing RuBisCO (chloroplast stroma). Crassulacean acid metabolism (CAM) plants keep their stomata (on the underside of the leaf) closed during the day, which conserves water but prevents the light-independent reactions (a.k.a. the Calvin Cycle) from taking place, since these reactions require CO2 to pass by gas exchange through these openings. Evaporation through the upper side of a leaf is prevented by a layer of wax. Genetic engineering Since RuBisCO is often rate-limiting for photosynthesis in plants, it may be possible to improve photosynthetic efficiency by modifying RuBisCO genes in plants to increase catalytic activity and/ or decrease oxygenation rates.

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 Unit III Part-B 1. Write notes on i). nod genes ii). nif genes iii). Hup genes iv). Bacteroid (Nov/Dec2013). i). nod genes: Rhizobium bacteria can invade the roots of leguminous plants and elicit the formation of root nodules. This process involves the expression of at least 20 nodule-specific genes encoded by the host plant, the so-called nodulin genes. ii). nif genes: The nif genes are genes encoding enzymes involved in the fixation of atmospheric nitrogen. The primary enzyme encoded by the nif genes is the nitrogenase complex which is in charge of converting atmospheric nitrogen- N2 To other nitrogen forms such as ammonia which the plant can use for various purposes. Besides the nitrogenase enzyme, the nif genes also encode a number of regulatory proteins involved in nitrogen fixation. iii). Hup genes: Hup genes are hydrogen uptaking genes, Diazotrophs evolve hydrogen at the time of N2 fixation , this H2 is having inhibitory effect on N2 fixation, this H2 will be rmoved by the action of hydrogenase enzyme. iv). Bacteroid: Inside the inner cortical region, rhizobium cells are enclosed in a membrane derived from the host cell and multiplied into 4- cells, finally become enlarged and pleuromorphic in nature called as Bacteroid . This may be swollen, star, irregular, club or y-shaped structures and is responsible for N2 fixation.

2. Give examples of NOD genes and their role ,how nodulation genes are isolated by complementation? Briefly write on plant specificity of rhizobium with example. (Nov. 2011). Nodulation (Nod) factors are signaling molecules produced by bacteria known as rhizobia during the initiation of nodules on the root of legumes. A symbiosis is formed when legumes take up the bacteria. The rhizobia produce nitrogen for the plant, and the legumes produce leghemoglobin to carry away any oxygen that would inhibit nitrogenase activity. 1. Nod factors 2. Nod gene expression 3. Rhizobial specificity.

3. How are nodulation genes isolated by gene complementation? Give examples of Nod gene products and their role. (Nov 2012). Nodulation (Nod) factors are signaling molecules produced by bacteria known as rhizobia during the initiation of nodules on the root of legumes. A symbiosis is formed when legumes take up the bacteria. The rhizobia produce nitrogen for the plant, and the legumes produce leghemoglobin to carry away any oxygen that would inhibit nitrogenase activity.  Nod factors  Nod gene expression  Rhizobial specificity

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4. Give a list of Nif genes, their function and explain the process of nitrogen fixation (Nov 2012). Explain by citing examples the genetic regulation of nitrogen fixation in the root nodules of leguminous plants. (May. 2014)

5. Elaborate on the genetic engineering of nodulation genes. (Nov. 2014) Genetic Engineering of Nodulation Genes: Establishment of nodules on the roots of leguminous plants is a prerequisite for nitrogen fixation. Certain genes involved in nodulation namely nod genes have been identified in Rhizobium melitoti. The technique of genetic complementation has been used to isolate nod genes from R. melitoti. A large number of nod genes (about 20 nodA — nodX) have been identified in diazotrophs. The nod genes are broadly divided into three groups: i. Common genes ii. Host-specific genes iii. Regulatory genes. The functions of each one of the nod genes in nodulation have not been clearly identified. Further, many more new nod genes are being discovered every year. Manipulation of nod genes? The process of nodulation is complex through the participation of a large number of nod genes, besides various other factors-concentration of nutrients, soil temperature, light, CO2 concentration etc. Despite attempts by several workers, no success has been reported to enhance the ability of Rhizobium sp for nodulation through genetic manipulations.

6. Discuss in detail about the different types of vectors used in plant gene transfer?(May-17) Co-integrate vector: In the co-integrate vector system, the disarmed and modified Ti plasmid combines with an intermediate cloning vector to produce a recombinant Ti plasmid (Fig. 49.5).

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Binary vector: The binary vector system consists of an Agrobacterium strain along with a disarmed Ti plasmid called vir helper plasmid (the entire T-DNA region including borders deleted while vir gene is retained). It may be noted that both of them are not physically linked (or integrated). A binary vector with T-DNA can replicate in E. coli and Agrobacterium. A diagrammatic representation of a typical binary vector system is depicted in Fig. 49.6. The binary vector has the following components.

Cauliflower mosaic virus (CaMV): CaMV infects many plants (e.g. members of Cruciferae, Datura) and can be easily transmitted, even mechanically. Another attractive feature of CaMV is that the infection is systemic, and large quantities of viruses are found in infected cells. A diagrammatic view of the CaMV genetic map is depicted in Fig. 49.8. The genome of CaMV consists of a 8 kb (8024 bp) relaxed but tightly packed circular DNA with six major and two minor coding regions. The genes II and VII are not essential for viral infection.

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Gemini Viruses as Vectors: The Gemini viruses are so named because they have geminate (Gemini literally means heavenly twins) morphological particles i.e. twin and paired capsid structures. These viruses are characterized by possessing one or two single-stranded circular DNAs (ss DNA). On replications, ss DNA forms an intermediate double-stranded DNA. The Gemini viruses can infect a wide range of crop plants (monocotyledons and dicotyledons) which attract plant biotechnologists to employ these viruses for gene transfer. Curly top virus (CTV) and maize streak virus (MSV) and bean golden mosaic virus (BGMV) are among the important Gemini viruses. It has been observed that a large number of replicative forms of a Gemini virus genome accumulate inside the nuclei of infected cells. The single-stranded genomic DNA replicates in the nucleus to form a double-stranded intermediate. Gemini virus vectors can be used to deliver, amplify and express foreign genes in several plants/ explants (, cultured cells). However, the serious drawback in employing Gemini viruses as vectors is that it is very difficult to introduce purified viral DNA into the plants. An alternate arrangement is to take the help of Agrobacterium and carry out gene transfer. RNA Plant Viruses as Vectors: There are mainly two type’s single-stranded RNA viruses: 1. Mono-partite viruses: These viruses are usually large and contain undivided genomes for all the genetic information e.g. tobacco mosaic virus (TMV). 2. Multipartite viruses: The genome in these viruses is divided into small RNAs which may be in the same particle or different particles, e.g. brome mosaic virus (BMV). HMV contains four RNAs divided between three particles. Plant RNA viruses, in general, are characterized by high level of gene expression, good efficiency to infect cells and spread to different tissues. But the major limitation to use them as vectors is the difficulty of joining RNA molecules . Use of cDNA for gene transfer: Complementary DNA (cDNA) copies of RNA viruses are prepared in vitro. The cDNA so generated can be used as a vector for gene transfer in plants. This approach is tedious and cumbersome. However, some success has been reported. A gene sequence encoding chloramphenicol resistance (enzyme- chloramphenicol acetyltransferase) has been inserted into brome mosaic virus genome. This gene expression, however, has been confined to protoplasts.

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 Unit IV Part-B

1. Write notes on the role of Agrobacterium mediated gene transfer. Describe in detail the Agrobacterium mediated genetic transformation in dicot plants. (May. 2014)

Agrobacterium is a gram negative bacteria used as a vector, introduction to A.rhizogens and A.tumifaciens , Ri and Ti plasmids Binary vectors – integration of two vectors based on its seq. homology and its uses Co-integrate vectors -Disarmed Ti plasmids cannot be used as a cloning vehicle because of larger size these plasmids are cloned into E.coli cells and gives an intermediate vector, this lack replication and conjugation , hence it is mixed with a helper plasmid using E.coli as aresult new vector called cointegrate is formed ex.pGV3850. 2. What are the molecular events that occur during Agrobacterium infection? Elucidate the process of DNA transfer and integration by Agrobacterium with suitable diagrams.(Nov 2012).(Nov/Dec2013)

Agrobacterium is a gram negative bacteria used as a vector, introduction to A.rhizogens and A.tumifaciens , Ri and Ti plasmids. The wounded plant part liberate phenolic compounds like acetosyringone from the root surface, which is sensed by the virD product, which induce the expression of various other vir genes. VirD2 product act as an nucleolytic enzyme and cleave the T-DNA strand, T-DNA single strand later on attached with E2 product enters into the plant cell through NLS and geteing integrated into the plant genome.

3. Write notes on various physical, chemical and biological gene transfer techniques. Elaborate the various method plant transformation methods and vectors available to engineer plants. Discuss their merits and demerits. (Nov. 2014) Various techniques are being used for gene transfer, directly the foreign gene along with the vector can be introduced into the plant through physical means like Lipofection, micro and macro injection, electrophoration and particle gun bombardment are used. Lipofection- using liposomes through endocytosis, since the DNA is coated with lipid layer nucleolytic enzymes can’t act on this. Chemically PEG can be used to enhace the DNA uptake of a protoplast. PEG at higher concentrations (12-25%) precipitate ionic macromolecules like DNA and stimulate their uptake by endocytosis. In biological methods, the foreign gene along with the vector can be introduced into the plant. Various vectors like plasmids, cosmids, phage, YAC, etc. can be used.

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4. Give an account on the role of various viral based vectors. Discuss the different types of viral vectors used in genetic engineering of plants and its applications. (May. 2014)

Many viruses or their isolated genomes are capable of infecting intact plant tissue. This made them suitable for use as plant transformation vectors. Salient features of viral vectors: 1. It easily introduce the genomic material into the intact plant cell or tissue by simple rubbing. 2. Infected cells express high level of the recombinant DNA. 3. Viral infection is systemic, hence it allow the transgene expression through out the plant. Various viral vectors are used for gene transfer, in which the unnecessary genes are replaced by the gene of our interest. Cauliflower mosaic virus is a type member of Caulimovirus, it contain dsDNA as genetic material and is divided, it infects wide range of dicots. The genome contain 8 tightly packed genes among this 2 regions are non essential this region can be replaced by the gene of interest,it is used as a viral vector for gene transfer Geminivirus is also used for plant gene transfer eg. Maize streak virus. 5. Describe the structure and properties of cauliflower mosaic virus and Gemini virus? (May 2012). Cauliflower mosaic virus is a type member of Caulimovirus, it contain dsDNA as genetic material and is divided, it infects wide range of dicots. The genome contain 8 tightly packed genes among this 2 regions are non essential this region can be replaced by the gene of interest,it is used as a viral vector for gene transfer Geminivirus is also used for plant gene transfer eg. Maize streak virus.( refer portal for more notes) 6. Describe how T – DNA is excised and transferred from agrobacterium and integrated into plant genome? (May 2012). Or Elucidate the process of DNA transfer and integration by Agrobacterium with suitable diagrams.(Nov 2012).

7. What are the modifications done on Ti plasmids for converting it to a transformation vector? Draw the map of a Agrobacterium binary vector (Nov 2012). Draw the structure of a binary vector and label the components and mention the function of each component. (Nov. 2014)

Plant transformation vectors are plasmids that have been specifically designed to facilitate the generation of transgenic plants. The most commonly used plant transformation vectors are termed binary vectors because of their ability to replicate in both E. coli, a common lab bacterium, and Agrobacterium tumefaciens, a bacterium

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 used to insert the recombinant (customized) DNA into plants. Plant Transformation vectors contain three key elements;  Plasmids Selection (creating a custom circular strand of DNA)  Plasmids Replication (so that it can be easily worked with)  T-DNA region (inserting the DNA into the agrobacteria) Consequences of the insertion 1. Foreign DNA inserted 2. Insertional mutagenesis (but not lethal for the plant cell – as the organism is diploid) Problem We want to transform the whole organism, not just one cell. This is done by transforming plant cells in culture, selecting transformed cells and regenerating an entire plant from the transformed cell (e.g. tobacco)

8. Write in detail about the Ti plasmid mediated gene transfer in plants? (May 17) Organization of Ti plasmid: The Ti plasmids (approximate size 200 kb each) exist as independent replicating circular DNA molecules within the Agrobacterium cells. The T-DNA (transferred DNA) is variable in length in the range of 12 to 24 kb, which depends on the bacterial strain from which Ti plasmids come. Nopaline strains of Ti plasmid have one T-DNA with length of 20 kb while octopine strains have two T-DNA regions referred to as TL and TR that are respectively 14 kb and 7 kb in length. A diagrammatic representation of a Ti plasmid is depicted in Fig. 49.3. The Ti plasmid has three important regions.

1. T-DNA region: This region has the genes for the biosynthesis of auxin (aux), cytokinin (cyt) and opine (ocs), and is flanked by left and right borders. These three genes-aux, cyto and ocs are referred to as oncogenes, as they are the determinants of the tumor phenotype. T-DNA borders — A set of 24 kb sequences present on either side (right and left) of T-DNA are also transferred to the plant cells. It is now clearly established that the right border is more critical for T-DNA transfer and tumori-genesis. 2. Virulence region: The genes responsible for the transfer of T-DNA into the host plant are located outside T-DNA and the region is referred to as vir or virulence region. Vir region codes for proteins involved in T-DNA transfer. At least nine vir-gene operons have been identified. These include vir A, vir G, vir B1, vir C1, vir D1, D2 and D4, and vir E1, and E2. 3. Opine catabolism region: This region codes for proteins involved in the uptake and metabolisms of opines. Besides the above three, there is ori region that is responsible for the origin of DNA replication which permits the Ti plasmid to be stably maintained in A. tumefaciens.

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19

Unit V Part-B 1. Write detailed notes on plant tissue culture and its importance. What are the various method of plant Tissue culture available? Discuss the applications of each one of them. (Nov. 2014)(May.2015).

Plant tissue : traditional methods of propagation, including:  The production of exact copies of plants that produce particularly good flowers, fruits, or have other desirable traits.  To quickly produce mature plants.  The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds.  The regeneration of whole plants from plant cells that have been genetically modified.  The production of plants in sterile containers that allows them to be moved with greatly reduced chances of transmitting diseases, pests, and pathogens.  The production of plants from seeds that otherwise have very low chances of germinating and growing, i.e.: orchids and nepenthes.  To clean particular plants of viral and other infections and to quickly multiply these plants as 'cleaned stock' for horticulture and agriculture. Plant tissue culture can be performed by the following phases.  Selection of media and standardization of media for tissue culture  Explant selection – root tip, shoot tip, etc.  Somatic embryogenesis – direct and indirect methods  Organogenesis - direct shoot or root development from the explant and  indirect shoot or root development via callus formation. 2. Write a detailed notes on the role of B.thuringiensis in the development of pest resistant plants. Describe the general structure of Bt toxin (cry protein) and list various cry proteins identified so far and exploited for pest resistance. (Nov 2012)./ How will you produce a BT resistant plant? Explain the process by citing an example. (May. 2014)

B. thuringiensis was first discovered in 1901 by Japanese biologist Shigetane Ishiwatari. In 1911, B. thuringiensis was rediscovered in Germany by Ernst Berliner, who isolated it as the cause of a disease called Schlaffsucht in flour moth caterpillars. In 1976, Robert A. Zakharyan reported the presence of a plasmid in a strain of B. thuringiensis and suggested the plasmid's involvement in endospore and crystal formation.[3][4] B. thuringiensis is closely related to B.cereus, a soil bacterium, and B.anthracis, the cause of anthrax: the three organisms differ mainly in their plasmids. Like other members of the genus, all three are aerobes capable of producing endospores.[1] Upon sporulation, B. thuringiensis forms crystals of proteinaceous insecticidal δ- endotoxins (called crystal proteins or Cry proteins), which are encoded by cry genes.[5] In most strains of B. thuringiensis, the cry genes are located on the plasmid.[6][7][8] Cry toxins have specific activities against insect species of the orders Lepidoptera (moths and butterflies), Diptera (flies and mosquitoes), Coleoptera (beetles), Hymenoptera (wasps, bees, ants and sawflies) and nematodes. Thus, B. thuringiensis serves as an important reservoir of Cry toxins for production of biological insecticides and insect-resistant genetically modified crops. When insects ingest toxin crystals, the alkaline pH of their digestive tract activates the toxin. Cry toxin gets inserted into the insect gut cell membrane, forming a pore. The pore results in cell lysis and eventual death of the insect. 3. What is molecular farming? Describe the oleosin system for hirudin and insulin production. (Nov. 2011).

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 Molecular farming (also known as molecular pharming or biopharming) is the use of genetically engineered crops to produce compounds with therapeutic value. These crops will become biological factories used to generate drugs and other difficult or expensive products. The term pharming can be used to describe plant derived pharmaceuticals, but it is more commonly used for products engineered in animals. Oleosin system for hirudin: A plant oleosin was used as a carrier for the production of the leech anticoagulant protein, hirudin (variant 2). The oleosin-hirudin fusion protein was expressed and accumulated in seeds. Seed- specific expression of the oleosin-hirudin fusion mRNA was directed via an Arabidopsis oleosin promoter. The fusion protein was correctly targeted to the oil body membrane and separated from the majority of other seed proteins by flotation centrifugation. Recombinant hirudin was localized to the surface of oil bodies as determined by immunofluorescent techniques. The oleosin-hirudin fusion protein accumulated to ca. 1% of the total seed protein. Hirudin was released from the surface of the oil bodies using endoprotease treatment. Recombinant hirudin was partially purified through anion exchange chromatography and reverse-phase chromatography. Hirudin activity, measured in anti- thrombin units (ATU), was observed in seed oil body extracts, but only after the proteolytic release of hirudin from its oleosin carrier . About 0.55 ATU per milligram of oil body protein was detected in cleaved oil body preparations. This activity demonstrated linear dose dependence. The oleosin fusion protein system provides a unique route for the large-scale production of recombinant proteins in plants, as well as an efficient process for purification of the desired polypeptide. Insulin production The anti-diabetic activity of insulin was first identified in 1921 and by 1951 the complete amino acid sequence had been determined. Since the standard source of the hormone was animal pancreas, a demand emerged for an alternative source of insulin that was safe, free of immunogenic contaminants and inexpensive. Human insulin was an attractive target for expression in microbes for it is a small polypeptide requiring only minimal post-translational processing to become functional. Expression in bacteria was successful and in 1982 it became the first recombinant protein to be approved for therapeutic use (Walsh, 1998). This released any restrictions on the amounts of insulin available, by producing a safe, active, recombinant human hormone at a low cost.

4. Give three categories of herbicides, their mode of action and strategy for herbicide tolerant plant development. (Nov. 2011)(Nov/Dec-2013)

Herbicide resistant Plants - plants with genes to synthesis the enzyme, which are denatured by herbicides, over expression of the specific genes will produce more and more amount of the enzyme this protects the plants against the herbicides ex. Glyposate resistance by introducing EPSP gene. Bromoxynil Resistance - In 1994, the European Union approved tobacco engineered to be resistant to the herbicide bromoxynil, making it the first commercially genetically engineered crop marketed in Europe. Glyphosate kills plants by interfering with the synthesis of the amino acids phenylalanine, tyrosine and tryptophan. It does this by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which catalyzes the reaction of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form 5-enolpyruvyl-shikimate-3-phosphate (ESP). ESP is subsequently dephosphorylated to chorismate, an essential precursor in plants for the aromatic amino acids: phenylalanine, tyrosine and tryptophan. These amino acids are used in protein synthesis and to produce secondary metabolites such as folates, ubiquinones and naphthoquinone. X-ray crystallographic studies of glyphosate and EPSPS show that glyphosate functions by occupying the binding site of the phosphoenolpyruvate, mimicking an intermediate state of the ternary enzyme substrates complex. Glufosinate or its ammonium salt DL-phosphinothricin is an active ingredient in several nonselective systemic herbicides - Basta, Rely, Finale, Ignite, Challenge and Liberty. It interferes with the biosynthetic pathway of the amino acid glutamine and with ammonia detoxification.

5. How can transgeneic be derived with non pathogen – derived resistance. (May 2012).

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 Pathogen Derived Resistance Protein Mediated Resistance Coat Protein Mediated Resistance Replicase Mediated Resistance Movement Protein Mediated Resistance RNA-mediated resistance sRNAs that target vRNAs for degradation Non-Pathogen, Protein Mediated Resistance Transcription regulators: TFs, and sZFPs DNA binding proteins Interferon-like strategies; ds-RNA degrading enzymes Translation initiation factors/co-factors

6. Describe the general structure of Bt toxin (cry protein) and list various cry proteins identified so far and exploited for pest resistance. (Nov 2012). Cry proteins are specifically toxic to the insect orders Lepidoptera, Coleoptera, Hymenoptera and Diptera, and also to nematodes. In contrast, Cyt toxins are mostly found in Bt strains active against Diptera. The Cry proteins comprise at least 50 subgroups with more than 200 members. Cry proteins are defined as: a parasporal inclusion protein from Bt that exhibits toxic effects to a target organism, or any protein that has obvious sequence similarity to a known Cry protein (Crickmore et al., 1998). Cyt toxins are included in this definition but it was agreed that proteins that are structurally related to Cyt toxins retain the mnemonic Cyt (Crickmore et al., 1998). Primary sequence identity among different gene sequences is the bases of the nomenclature of Cry and Cyt proteins. Additionally, other insecticidal proteins that are not related phylogenetically to the three-domain Cry family have been identified. Among these, are binary-like toxins and Mtx-like toxins related to B. sphaericus toxins, and parasporins produced by B. thuringiensis (Crickmore et al., 1998).

Three dimensional structures of insecticidal toxins produced by Bacillus thuringiensis Cry1Aa, Cry2Aa, Cry3Aa, Cry3Bb, Cry4Aa, Cry4Bb and Cyt2A. Cyt proteins, on the other hand, have a single α-β domain comprising of two outer layers of α-helix hairpins wrapped around a β-sheet (Li et al., 1996, Fig 1). Cyt toxin is structurally related to volvatoxin A2, a PFT cardiotoxin produced by a straw mushroom Volvariella volvacea (Lin et al., 2004).

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BT 6010 - Plant Biotechnology Department :Biotechnology 2018- 19 7. How are therapeutically important coumpounds produced in plants on a large scale? Discuss the merits and demerits of using plant system (Nov 2012). Explain how plants can be used as a bioreactor for producing therapeutics simultaneously discuss the limitations also. (Nov. 2014)(May.2015).

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