This document discusses two case studies involving genetically modified crops: 1) Drought tolerant transgenic plants developed using genes for abiotic stress tolerance through genetic engineering. Genes used include structural genes for late embryogenesis abundant (LEA) proteins and regulatory genes for key enzymes. This allows improved water stress management compared to conventional breeding. 2) Genetically engineered potatoes (Innate) modified using RNA interference to suppress expression of certain genes to reduce browning and increase resistance to bruising and soft rot. This helps improve potato quality and shelf life.

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
 pBR322
 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. 1953-Watson & Crick -Discovery of DNA structure
1969- Herbert Boyer -Isolated restriction enzyme EcoRI from
E.coli
1970- Temin & Baltimore- Discovered the reverse
transcriptase enzyme from retrovirus
1972- Paul berg, David Jackson & Robert Symons
Successfully generated rDNA molecules
1973 – S.cohen and H.Boyer- Developed a recombinant
Plasmid
1975 – Edwin Southern - Developed a method for detection of
Specific DNA fragments called Southern blotting Technique
History of Recombinant DNA Technology

7. Procedure for recombinant DNA technology
1. Isolation of a desired gene
2. Insertion of the isolated gene in a suitable
vector to obtain recombinant DNA
3. Introduction of the recombinant DNA into
suitable host
4. Selection of the transformed host cells i.e.,
identification of the clone containing desired
gene
5. 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

10. 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:
1) Restriction endonuclease digestion of genomic DNA
2) Mechanical shearing of genomic DNA
3) cDNA duplexes from RNA transcripts
4) Direct chemical synthesis
5) Polymerase chain reaction

11. 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

12. Type I Type II Type III
 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

14.  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

15.  Most type II endonucleases cleave the DNA molecules with their specific recognition
sequences but some produce cuts immediately outside the target sequence
Eg: NlaIII,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.

16. 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 37oC but it is often used at much lower temperatures(4-
15oC) to prevent thermal denaturation of short base paired regions

17. cDNA
 cDNA 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.

18.  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
i. Ideally they should be fairly small molecules to facilitate isolation
ii. 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
iii. It is desirable to have some markers that will enable the vector to be detected
iv. 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:

19.  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:

20.  pbr322 is most popular and widely used plasmid
vector of 4,363 bp
 The name pBR 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: pBR325, pBR327
 It is created in 1977 in the laboratory of Herbert boyer at the
university of california,sanfranscisco
 It 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
pBR322
Dr.Francisco Bolívar Zapata

21.  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 CaCl2 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 37oc for 60-90 min to
enable plasmids to become established and permits the expression of traits.
Introduction of Recombinant DNA into a suitable host

22. 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.

23.  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

24.  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:
1. Target DNA
2. Two Nucleotide primers
3. dNTPs – deoxynucleotriphosphates
4. Thermostable DNA Polymerase (Taq Polymerase)
5. Mg+2 ions
6. Buffer solution
Polymerase Chain Reaction - PCR

25. Procedure for
PCR:
It involves mainly
3steps:
I. Denaturation
II. Annealing
III. Primer Extension
Denaturation:
 The reaction mixture is
first heated to a
temperature between 90-
98 oC that ensures DNA
denaturation
 The Duration of this cycle
is usually 2-3 minutes
Annealing:
 The mixture is now cooled
to a temperature 40-60oC
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 72oC
 Best results are obtained
when the period of
extension kept at the rate
of 1min per kb
Munagala Sai
Kiran18MSCGPB090

26.  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.
Eg: plasmid pBR322 contains genes for ampicillin resistance Apr and tetracycline
resistance Tcr 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

27. 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

28.  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

29. 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

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

31. 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

32. 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

33. 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

34. 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 asparagine 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

36.  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.

38. Comparison between Normal potato and Innate Potatoes after frying

39. Crop development

40. Potato commercialization

41.  Desmod S.T.Nicholl-An intoduction to Genetic engineering 3rd
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

42. Prepared & Presented by:
Munagala Sai Kiran
18MSCGPB090
M.Sc.Ag.(Genetics and Plant breeding)
Department of Genetics and Plant breeding
SHUATS
skrn.108@gmail.com

43. Munagala Sai Kiran
18MSCGPB090