Recombinant DNA technology for Crop improvement

A recombinant DNA molecule is produced by joining together two or more DNA segments usually originating from two different organisms.
More Specifically, a recombinant DNA molecule is a vector into which desired DNA fragment has been inserted to enable its cloning in an appropriate host.
Recombinant DNA molecules are produced with one of the following objectives:
1. To obtain large number of copies of specific DNA fragments.
2. Large scale production of the protein encoded by the gene.
3. Integration of the desired DNA fragment into target organism where it expresses itself.

Drought tolerant-genetically modified plants:
Present abiotic stress is a major challenge in our quest for sustainable food production as these may reduce the potential yields by 70% in crop plants
Of all abiotic stress, drought is regarded as the most damaging
Transgenic plants carrying genes for abiotic stress tolerance are being developed for water stress management
Conventional breeding approaches, involving inter specific and inter generic hybridizations and mutagenesis have been limited success.
Major problems have been the complexity of drought tolerance & low genetic yield components under drought conditions.
Unlike conventional plant breeding there is no need of repeated back crossing
Gene pyramiding or gene stacking through co-transformation of different genes with similar effects can be achieved.

1. Recombinant DNA Technology for Crop Improvement - Drought tolerant-genetically modified plants & Genetically engineered Potatoes (Innate) Munagala Sai Kiran 18MSCGPB090

2. GPB-880 Masters Seminar On Recombinant DNA Technology for Crop Improvement - Drought tolerant-genetically modified plants & Genetically engineered Potatoes (Innate) Presented by: Munagala Sai Kiran 18MSCGPB090 M.Sc.Ag .(Genetics and Plant breeding) Department of Genetics and Plant breeding

3. Contents: Introduction Recombinant DNA Technology History Procedure for rDNA technology Isolation of DNA & generation of fragments Restriction endonucleases & Types Recognition sequences Cleavage patterns DNA ligase p BR322 PCR Case study 1: Drought stress tolerant Transgenics Case study 2: Genetically engineered potato

4. Introduction Natural Recombination of genes occur during meiotic crossing over which results in appearance of undesirable genes along with desired. In contrast genetic engineering is novel and desired combination of genes through recombinant DNA technology. There is a serious limitation on the movement of genes across taxonomic borders. However, during evolution many bacterial genes have been directly integrated into the human genome.

5. Recombinant DNA Technology A recombinant DNA molecule is produced by joining together two or more DNA segments usually originating from two different organisms. More Specifically, a recombinant DNA molecule is a vector into which desired DNA fragment has been inserted to enable its cloning in an appropriate host. Recombinant DNA molecules are produced with one of the following objectives: 1. To obtain large number of copies of specific DNA fragments. 2. Large scale production of the protein encoded by the gene. 3. Integration of the desired DNA fragment into target organism where it expresses itself.

6. History of Recombinant DNA Technology

7. Procedure for recombinant DNA technology Isolation of a desired gene Insertion of the isolated gene in a suitable vector to obtain recombinant DNA Introduction of the recombinant DNA into suitable host Selection of the transformed host cells i.e., identification of the clone containing desired gene Multiplication/expression of the introduced gene in the host

8. Isolation of DNA The first step in any isolation protocol is disruption of the cell wall that eventually opens the cell as gentle as possible. Preferably utilizing enzymatic degradation of cell wall material and detergent lysis of cell membranes. Following the cell disruption most methods involve a deproteinisition stage. This can be achieved by one or more extractions using phenol/Chloroform mixtures. Followed by centrifugation the nucleic acids remain mostly in the upper aqueous phase. Finally, ice-cold alcohol (either ethanol or isopropanol) is carefully added to the DNA sample. DNA Precipitate

9. Generation of DNA fragments for cloning The identification and isolation of the desired gene or DNA fragment is a critical step in r-DNA technology The DNA fragments used for cloning are generally obtained as follows: Restriction endonuclease digestion of genomic DNA Mechanical shearing of genomic DNA cDNA duplexes from RNA transcripts Direct chemical synthesis Polymerase chain reaction

10. Restriction Endonucleases Endonucleases are enzymes that produce internal cuts called cleavage in DNA molecules. The class of endonucleases cleaves DNA near those sites which have specific base sequences such endonucleases are known as restriction endonucleases. The sites which are recognised by them are called recognition sequences. These enzymes are found in bacterial cells where they function as part of protective mechanism called the restriction-modification system. In this system the restriction enzyme hydrolyses any exogenous DNA that appears in the cell. To prevent the enzyme acting on the host cell DNA a methylase enzyme modifies the host DNA by methylation of particular bases in the restriction enzymes recognition sequence. This prevents the enzyme from cutting the DNA

11. Type I are Complex endonucleases. they have recognition sequences of 15bp. They cleave the DNA about 1000 away from 5’-end. Type II are Remarkably stable There are more than 350 different type II endonucleases with over 100 different recognition sequences. They require Mg +2 Ions for cleavage. Type III are intermediate between the Type I and Type II enzymes. The recognition sequences up to 20bp away. Type I and Type III restriction enzymes are not used in gene cloning The Type III enzymes recognize asymmetric target sites Type I enzymes are complex & Cleave away from the target DNA sites

12. The recognition sequences for Type II endonucleases form palindromes with rotational symmetry. In a palindrome, the base sequence in the second half of a DNA strand is the mirror image of the sequence in the first half. But in a palindrome with rotational symmetry the base sequence in the first half of one strand of a DNA double helix is the mirror image of the second half of its complementary strand. Most of the type II restriction endonucleases have recognition sites of 4,5 or 6 bp , which are predominantly GC rich Recognition Sequences

13. Most type II endonucleases cleave the DNA molecules with their specific recognition sequences but some produce cuts immediately outside the target sequence Eg: Nla III,Sau The cuts are two types 1) Staggered 2) Blunt ends Two protruding ends generated by such cleavage by a given enzyme have complementary sequences as a result they readily pair with each other under annealing conditions. Some restriction enzymes on the other hand cut both the strands of a DNA molecule at the same site resulting in terminal or Blunt ends Cleavage Patterns Most enzymes produce staggered cuts in which two strands of a DNA double helix are cleaved at different locations this generates protruding ends.

14. DNA ligase –Joining DNA molecules DNA ligase is an important cellular enzyme as its function is to repair broken phosphodiester bonds . The enzyme used most often in experiments is T4 DNA ligase, which is purified from E.coli cells infected with bacteriophage T4 The enzyme works best at 37 o C but it is often used at much lower temperatures(4-15 o C) to prevent thermal denaturation of short base paired regions

15. c DNA c DNA or Complementry DNA produced by using mRNA as a template. DNA copy of an RNA is produced by enzyme reverse transcriptase generally obtained from avian myeloblastosis virus(AMV). This enzyme performs similar reactions as DNA polymerase and has absolute primer requirement with a free 3’-OH. The reverse transcriptase extends the 3’-end of the primer using mRNA molecule as a template. This process produces the RNA DNA hybrid molecule, the DNA strand is obviously the DNA copy( cDNA ) of mRNA strand. The RNA strand is digested either by Rnase or by alkaline hydrolysis this frees the single stranded cDNA The end of this cDNA serves as its own primer and provides the free 3’-OH required for the synthesis of its own complementery strand.

16. A vector is a DNA molecule that has ability to replicate autonomously in an appropriate host cell There are certain features that vectors must posses Ideally they should be fairly small molecules to facilitate isolation There must be origin of Replication so that DNA can be copied and thus maintained in the cell population as the host organism grows and divides It is desirable to have some markers that will enable the vector to be detected The vector must also have must also have atleast one unique restriction endonuclease recognition site to enable DNA to be inserted during production of recombinants. Plasmids have these features and are extensively used as vectors in cloning experiments Vector:

17. Plasmids are extra chromosomal,double stranded,circular,self -replicating DNA molecules. Plasmids are dispesable,they often confer some traits.(such as Anti-biotic resistance) Bacteriophages are the viruses replicate within the bacteria They are literally ‘eaters of bacteria’ – viruses that depend upon bacteria for their propagation. In case of certain pahges their DNA gets incorporated into bacterial chromosome. Cosmids are the engineered vectors that combine charcteristics of both plamids and phage They carry larger fragments of foreign DNA compared to plasmids This include Yeast artificial chromosome, Human artificial chromosome and Bacterial artificial chromosome They can accept large fragments of foreign DNA Plasmids : Bacteriophages: Cosmids : Artificial Chromosome vectors:

18. p br322 is most popular and widely used plasmid vector of 4,363 bp The name p BR p signifies plasmid is boliver and R is from Rodriguez The numerical 322 distinguishes this plasmid from other Plasmids developed in the same laboratory Eg: p BR325, p BR327 It is created in 1977 in the laboratory of Herbert boyer at the university of california,sanfranscisco I t permits plasmid replication even when chromosome replication and cell division are inhibited by amino acid starvation or Chloromophemicol It has selectable markers (tetracycline and ampicillin) single or unique recognition sites for 12 different restriction enzymes p BR322 Dr.Francisco Bolívar Zapata

19. The techniques of transformation and transfection repents the simplest methods available for getting the recombinant DNA into cells. Transformation refers to the uptake of the plasmid DNA where as Transfection is uptake of the phage DNA. Transformation: Transformation of E.coli cells is the one of the classic techniques of Gene manipulation. Transformation in bacteria was demonstrated in Fredrick Griffith in 1928. Transformation can be achieved by treating the cells with the ice cold CaCl 2 this makes cells competent. Then the rDNA is mixed with the cells incubating on ice for 20-30 minutes. Then giving a brief heat shock enables the entry of DNA into cells. The transformed cells are usually incubated in a nutrient broth at 37 o c for 60-90 min to enable plasmids to become established and permits the expression of traits. Introduction of Recombinant DNA into a suitable host

20. Electroporation: This is based on the principle of High voltage electric pulses can induce cell plasma membranes to fuse. This increases the permeability of the cell wall. Liposome-mediated gene transfer( Lipofection ) is mediated by the lipid molecules.

21. Plant cells pose the problem with rigid cell wall, which is barrier to DNA uptake . This can be alleviated by the production of protoplasts in which is cell wall is removed enzymatically. The protoplasts can be transformed using a technique such as electroporation. Alternative methods of DNA transfer Micro injection and Particle Bombardment method: Micro injection is use very fine needle and inject the DNA directly into the nucleus This technique is called micro injection. The cell is held on the glass tube by mild suction and the needle used to pierce the membrane. Where as in the particle bombardment technique involves literally shooting DNA into cells The DNA is used to coat microscopic tungsten particles known as micro projectiles then accelerated on a macroprojectile by firing by using compressed gas to drive the particles This is called Biolistic DNA delivery

22. The PCR technique is developed by Kary mullis in1985 He was awarded nobel prize in the year 1993 in field of chemistry for his discovery of PCR PCR generates microgram(µg) quamtities of DNA copies of the desired DNA or RNS segment, present even as a single copy in the initial preparation in a matter of few hours The PCR is carried invitro utilizes following: Target DNA Two Nucleotide primers dNTPs – deoxynucleotriphosphates Thermostable DNA Polymerase ( Taq Polymerase) Mg +2 ions Buffer solution Polymerase Chain Reaction - PCR

23. Procedure for PCR : It involves mainly 3steps: Denaturation Annealing Primer Extension Denaturation : The reaction mixture is first heated to a temperature between 90-98 o C that ensures DNA denaturation The Duration of this cycle is usually 2-3 minutes Annealing: The mixture is now cooled to a temperature 40-60 o C permits annealing of the primer to the complementary sequences in the DNA The duration of annealing step in usually 1min during the first and subsequent cycles of PCR Primer Extension: The primers are extended towards each other so that the DNA segment lying between the two primers copied During primer extension is usually 2min at 72 o C Best results are obtained when the period of extension kept at the rate of 1min per kb Munagala Sai Kiran18MSCGPB090

24. Genetic selection and screening methods rely on the expression or non-expression of certain traits. Usually these traits are encoded by the vector but some cases it may be due to the target DNA. One of the simplest genetic selection methods involves the use of antibiotics to select for the presence of vector molecules. E g: plasmid pBR322 contains genes for ampicillin resistance Ap r and tetracycline resistance Tc r thus presence of plasmid can be detected by placing the recombinants on the agar medium that containing these chemicals. Insertional inactivation: The presence of cloned DNA fragments can be detected if the insert interrupts the coding sequence of a gene this approach is known as insertional inactivation Genetic selection and Screening of Recombinants

25. Case study -1 Drought Stress tolerance Transgenics Based on the Research paper Published by: Satbir S. gosal,Shabir H. wani and Manjit S. Kang Satbir S. gosal,Shabir H. wani and Manjit S. Kang Biotechnology and Drought Tolerance Journal of Crop Improvement , 23:19–54, 2009 Institution : School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana . & Drought tolerance in crop plants American Journal of Plant Physiology 5(5): 241-256,2010

26. Present abiotic stress is a major challenge in our quest for sustainable food production as these may reduce the potential yields by 70% in crop plants Of all abiotic stress, drought is regarded as the most damaging Transgenic plants carrying genes for abiotic stress tolerance are being developed for water stress management Conventional breeding approaches, involving inter specific and inter generic hybridizations and mutagenesis have been limited success. Major problems have been the complexity of drought tolerance & low genetic yield components under drought conditions. Unlike conventional plant breeding there is no need of repeated back crossing Gene pyramiding or gene stacking through co-transformation of different genes with similar effects can be achieved. Drought stress-tolerance transgenics

27. Types of Genes used for Developing abiotic stress Resistance through Genetic engineering Transgenic plants carrying genes for abiotic stress tolerance are being developed for water management There are two types of Genes 1) Structural Genes 2) Regulatory Genes For Stress induced LEA proteins For key enzymes for biosynthesizing osmolytes For Redox Proteins and Detoxifying enzymes Structural Genes Proline Glycinebetaine Mannitol Trehalose

28. Regulatory genes Dehydration responsive element binding factors Zinc finger proteins Transcriptional factor genes NAC,NAM,ATAF & CUC Proline cycle

29. Mechanisim Trasngenes Plant species Trasnformation Method Promoter Remarks Proline P5CS (Pyrroline-5-carboxylate synthetase ) from vigna aconitiflora Nicotiana tabacum Agrobacterium CaMV 35S Transgenic plants produced 10-18 more proline than control plants. Over production of proline also enhanced root biomass and flower development P5CS from vigna aconitiflora Saccharum officinarum Particle gun AIPC Stress indcucible proline accumulation in transgenic sugar cane plants under water- deficit stress acts as a cpmponent of antioxidative defence sysytem Genetic engineering of Crop plants for Abiotic stress tolerance

30. Mechanisim Trasngenes Plant species Trasnformation Method Promoter Remarks Glycinebetaine CMO (choline monooxygenase ) Oryza sativa Agrobacterium Maize ubi Transgenic plants were tolerant to salt and temperature stress at seeding stage Mannitol mtlD from E.coli Triticum aestivum Particle gun Maize ubi Tolerance to water stress and Salinity LEA Protiens HVA 1 from Hordeum vulgare Oryza sativa Particle Rice actin 1 Second generation transgenic plants shown improved growth characteristics Regulatory genes CBF1 from A.thaliana Lycopersicon esculentum Agrobacterium CaMV 35S Transgenic tomato plants were more resistant to water –deficit stress than the wild types DREB 1A from A.thaliana Arachis hypogea Agrobacterium CaMV 35S rd29A Transgenic plants show increased transpiration efficiency an important feature

31. Case study -2 Genetically Engineered Potato Based on the Research Article Published by: Haven Baker Bringing Biotech Potatoes to Market J.R. Simplot Company Boise, Idah

32. Innate The genetically modified Innate potato was approved by the United States Department of Agriculture in 2014 The cultivar was developed by J. R. Simplot Company. It is designed to resist black spot bruising, browning and to contain less of the amino acid  aspa ragine   that turns into  acrylamide  during the frying of potatoes Acrylamide is a probable human carcinogen, so reduced levels of it in fried potato foods is desirable Ranger russet Russet bur bank

33. Four varieties have been improved via Innate 1.0: Russet Burbank, Ranger Russet,Atlantic and Snowden . Russet Burbank and Russet Ranger are the primary French-fry varieties. Atlantics and Snow are the primary varieties used to make chips. improvements result in benefits to growers, processors and consumers. Browning occurs not only after cutting but also when stacked in storage which causes pressure bruising. Innate Atlantic 2.0 lines Y9 and Y15 fry with reasonably good colour after six months of storage at 38°F.

34. Comparison between Normal potato and Innate Potatoes after frying

35. Crop development

36. Potato commercialization

37. Desmod S.T.Nicholl -An intoduction to Genetic engineering 3 rd edition,Cambridge University Press. Singh.B.D – Biotechnology Expanding Horizons,Kalyani Publishers. Satbir S. gosal,Shabir H. wani and Manjit S. Kang Biotechnology and Drought Tolerance Journal of Crop Improvement , 23:19–54, 2009. Haven Baker Bringing Biotech Potatoes to Market J.R . Simplot Company Boise, Idah . Drought tolerance in crop plants American Journal of Plant Physiology5(5 ): 241-256,2010 . References: Munagala Sai Kiran 18MSCGPB090

38. Prepared & Presented by: Munagala Sai Kiran 18MSCGPB090 M.Sc.Ag .(Genetics and Plant breeding) Department of Genetics and Plant breeding SHUATS s [email protected]

39. Munagala Sai Kiran 18MSCGPB090