ant DNA Tech

8/6/2019 ant DNA Tech

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Maneesh Mishra, Ph.D.Maneesh Mishra, Ph.D.

Biotechnology LaboratoryBiotechnology Laboratory

Central Institute for Subtropical HorticultureCentral Institute for Subtropical Horticulture

LucknowLucknow-- 227 107227 107



Increase in Crop Yield

Green Revolution

Hybrids (Selective breeding Among

Sexually Compatible Partners)Use of Chemical Fertilizers

GREEN REVOLUTION



TRADITIONAL BREEDING

teosinte

cob

corn

Wild tomato

(Lycopersicon pimpinellifolium )

D= 1 cm





For years, framers have been selecting for certainfeatures in crops producing plants that are easierto grow, tastier, and bigger without knowing the

exact mechanism of how this occurs.

Nowadays scientists can transfer genesfrom one organism to another unrelatedorganism, producing what is now knownas genetically modified organism or transgenic animal/plantµ.





DNA in chromosomes carries biological

information



�� GenesGenes = the coding system for instructions

�� A g ene A g ene = is a segment of DNA

Guanine

(G)

Cytosine

(C)

Adenine

(A)

Thymine

(T)

bases DNA

DNA and GenesDNA and Genes

gene



Genes are made of DNA

Promoter Protein coding region

Protein

Gene encode proteins. Proteins performs the biological functions

determining the characteristics of organism



A gene is a DNA segment that encodes

a specific protein that contributes to

expression of a trait.

�The steps of the process:

mRNA molecule is transcribed accordingto the message encoded in the DNAcoding sequence (i.e., gene).

A protein is translated from the mRNAmolecule.

the production of this protein in the cellwill result in an expression of a trait.



Genes encoding important traits

Extended shelf life

Insect, disease resistance

Uniform ripening,Increased sugars

Flavors

Antioxidants

Abiotic stress

Gene IdentificationGene Identification



Gene manipulation

Genes are manipulated in many ways

Suppression ( a deleterious gene)

Over expression ( favorable gene)

Modification (enhancing new characteristics)

Transgene expression ( introducing new functions)



How Genetic engineering technology works

A gene of known function (gene controlling fruit

ripening) is identified and isolated.

The gene must be stably incorporated in to plant.

The gene must be heritable.



Marker gene Promoter TransgeneTermination

sequence

SelectionWhere

When

How much

What Stop

A DNA construct is used for transformationA DNA construct is used for transformation



Genes are introduced in the plants using bacterial plasmid Genes are introduced in the plants using bacterial plasmid

as vectorsas vectors

Plasmids are circular molecules of DNA found in bacterial cell. They

confer survival of antibiotics



How to get DNA in to plantsHow to get DNA in to plants

Agrobact erium

Particle bombardment

Electroporation



Agrobact erium Agrobact erium tumef aci enstumef aci ens isis aa

naturallynaturally occurringoccurring bacteriumbacterium

thatthat hashas aa abilityability toto transfertransfer thethe

piecepiece of of DNADNA inin toto chromosomechromosome

TheThe abilityability hashas beenbeen usedused toto

transfertransfer DNADNA oror transformtransform plantsplants



TTRANSFOR MATIONRANSFOR MATION



Particle Bombardment System

R upture disc

Plant material

Stopping plate

R upture disc

Gold particle

Plant material

Stopping plate



³Gene Gun´ Technique³Gene Gun´ Technique

DNA coatedgolden particles

Gene gun

Cell division

A plant cell with

the new gene

Transgenic plant

Plant cell

Cell¶s DNA



G

EN

E

G

U

N



CaMV Promoter OCS terminator

Bam H 1 Xba 1

CaMV PromoterNOS terminator

Bam H 1 Xba 1

pBinAR -12.2 Kb in sense orientatio

pBI121(13.5 Kb) in antisense orientation

GENE CONSTR UCT FOR PR SV



P

R

O

C

E

S

S



S

E

L

E

C

T

I

O

N



83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

First transgenic plant

First Bt corn plants

First field tests

Delay-ripening tomato Commercialized in the US

Rotting resistant tomato approved by FDA

Herbicide resistant, insect resistant plants commercialized

GM maize approved by EU

HISTORY

OF TRANSGENIC

PLANTS



Transgenic Fruit crops



Transgenic plum

Nontransgenic plum

Plum Pox Virus



Flavr Saver Tomato: Extended shelf lifeFlavr Saver Tomato: Extended shelf life



Transgenic tomatoes with

regulated ethylene production.

Delayed harvesting.

Fruits stays longer in the field.

Non-transgenic Transgenic tomato

Tomato with 4 with 2 colours

colours

g



Tomato with reduced polygalucturoseTomato with reduced polygalucturose

Increased TSS in pasteIncreased TSS in paste



Tomato plants artificially

inoculated with bacterial

speck disease

Plants on left has been genetically engineered

with gene for resistance to disease and plant onright has not.



Transgenic tomato growing against salt stress

Transgenic grown

at x 40 salt

Non

transgenicgrown at x 40

salt

Non-

transgenic

grown at

200 mM

NaCl

Non-

transgenicgrown at 5

mMNaCl



Delayed softening/ripening in transgenic apple

Non-Transgenic Transgenic







Transgenic crops will create monsterTransgenic crops will create monster



Transgenic cropsTransgenic crops--a curea cure--allall



Many things are not natural. Good things result fromMany things are not natural. Good things result from

experimentation and improvement of nature:experimentation and improvement of nature:AntibioticsAntibiotics

AnaestheticsAnaesthetics

Warm housesWarm houses

And now, more nutritious foods!And now, more nutritious foods!

The testing of GM

foods is exemplary in its detail and takes atThe testing of GM

foods is exemplary in its detail and takes atleast four years of testing for it to be introduced to consumers.least four years of testing for it to be introduced to consumers.

GMOs have actually reduced the use of herbicides andGMOs have actually reduced the use of herbicides andpesticides.pesticides.

Now, is the time to experiment and not when a natural disaster Now, is the time to experiment and not when a natural disaster (Ex. overpopulation) plagues us and it is too late.(Ex. overpopulation) plagues us and it is too late.

TR UTH IS





GENES IN THE SALAD

In the form of salad on an average we

eat: 100,000,000,000,000 (100 billion

genes)

(500 g Mixed Salad, Plant Cell: 50µm, Density:1g/cm,

~4x109 cells, 25,000 genes per cell)



ThroughoutThroughout history,history, newnew scientificscientificdiscoveriesdiscoveries havehave challengedchallenged

conventionalconventional thinkingthinking (Galileo(Galileo--

CopernicusCopernicus theory,theory, SunSun--EarthEarth

Movement)Movement) However,However, over over thethe yearsyearssocietysociety hashas learnedlearned toto acceptaccept indeed,indeed,

celebratecelebrate thesethese discoveriesdiscoveries andand socialsocial

andand intellectualintellectual benefitsbenefits theythey havehaveconveyedconveyed..



Transgenic cropsTransgenic crops--A bountifulA bountiful

harvestharvest



± Patents, plant breeder¶s rights, tr ade secrets, tr ade

marks, copy rights

± Gene constructs would be covered by patents

IPR in Biotechnology



A gene construct is built from

genetic material isolated from

different organisms

± E.g. promoter from CaMV, a bacterial DNA vector

( Agrobacterium plasmid), one or more genes th

atmay have been modified in the labor atory,

termination and signalling sequences and a

selectable marker gene.

� Involves both Process and Product

Gene Construct



An exclusive right gr anted for an invention,

which is a product or a process that

provides, in gener al, a new way of doing

something, or offers a new technical

solution to a problem.

----------------------------------------------------------------

Once issued, a patent gives the inventor the legal right to create a

limited monopoly by excluding others from creating, producing,

selling or importing the invention.

This right is of limited dur ation, for a minimum period of 20 years

Patent



Usefulness or industrial application

Newness or novelty

Non-obviousness or inventive step

Conditions for granting Patents



Be careful while obtaining permission to

use the constructs for research

N ote

The high incidence of the use of the promoter CaMV /35S

The use of genes with very specific characteristics

like the Cry genes

The use of selectable markers such as GUS,

kanamycin and hygromycin resistance genes. The basic constructs containing selectable markers,

promoters, and genetic markers

Patent vis a vis Gene

Constructs



Be aware

Of the principles of territoriality of patents, and, as a consequence, of the

implications for the application of

their research.

Of the fact that whether a gene

construct is patented or not and if

patented, about the need to seek thelicense.



Pl ease note

Gene constructs may not be patentedin our country, and therefore the

original patent may not be important

for the academic research purpose.

However, the actual implications

depend on the final use of the

research results e.g. a transformed

plant.



Mostly the gene constructs are notobtained by a formal contractual

arrangement ("lacking written

approval", "international collaboration",

and "not known").

Furthermore, the use of licenses is low,

in contrast to the use of non-writtenagreements and international

collaboration.



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