This document provides an overview of genetic engineering and transgenic breeding techniques. It discusses cell culture and transformation methods like somatic embryogenesis and organogenesis to obtain transgenic plants. Methods of plant transformation include direct transformation, biological delivery methods, and selecting transgenic events. Molecular biology techniques for plant transformation involve overexpression vectors, promoters for transgene expression, RNA interference, and zinc-finger nucleases. Transgenic breeding involves transferring transgenes into elite lines through backcross breeding over multiple generations. Examples are given of transgenic crops developed for increased yield, insect resistance, disease resistance, herbicide tolerance, drought tolerance, and altering morphological characteristics.

1. PRESENTED BY: Pawan Nagar
Reg. no.: 04-2690-2015
M.Sc. (Fruit Science)
Genetic engineering &
Transgenic breeding

2. Introduction
Cell culture and transformation
Cell Culture and Somatic Embryogenesis, a Means to Obtain
Transgenic Plants
o1 .Somatic Embryogenesis
o2 .Organogenesis
Methods of Plant Transformation
o1 .Direct Transformation
o2. Biological Delivery Methods
o3. Selecting Transgenic Events
Molecular Biology for Plant Transformation
o1. Overexpression Vectors
o2. Promoters for the Expression of Transgenes
o3. RNA Interference
o4. Zinc-Finger Nucleases
Breeding with Transgenics / Transgenic breeding
CONTENT

3. What is Transgenic Breeding ?
Genetic improvement of crop plants, domestic animals,
microbes through Biotechnology.
What is Transgenics ?
A genotype developed by the process of genetic
engineering.
Or
A genotype containing foreign gene or modified gene of
different species transferred by process of genetic engineering.
What is Transgene ?
Foreign gene or modified gene of any species which is
used for development of transgenics. These may be from
related wild species, microbes (Bacteria, viruses and fungi) or
unrelated species.

4. Fig 1: A diagram re-drawn from Harlan and de Wet (1971) to include a description of gene
pools as they relate to the use of transgenes . Circles are representations of the
primary- (GP-1), secondary- (GP-2), tertiary- (GP-3), and quaternary-gene pools (GP-4)

5. Why Create Transgenic Plants?
1. Improve agricultural value of plant
increase yield (herbicide-resistance, pest-resistance)
enhance nutrition
enhance taste
2. Plants can produce proteins for human needs (pharmaceutics)
3. Modified plants can be used to study effects of genes
An entire plant can be regenerated from a single cell
***TOTIPOTENCY***
No separation of germ and somatic cells

6. Overview of The Process
 There are five major steps involved in genetically engineering
plants. These are DNA isolation, single gene cloning, gene
designing, cell transformation, and backcross breeding.
• DNA is extracted from an organism that has the desired trait.
• The desired gene is located and copied.
• The gene is inserted into a single plant cell using a transformation
method. If the transgene successfully lands in the cells nucleus and
is incorporated into one of the chromosomes, then the trait that it
codes for will be expressed in the cell's offspring.
• The cell multiplies and grows a new plant that contains the
transgene in all of its cells.
• Through backcross breeding the transgenic plant is crossed with a
plant from a high yielding line. The resulting hybrids are the
genetically modified plants that can enter the marketplace.

7. General procedure of recombinant DNA technology

8. Cell culture and transformation
Cell Culture and Somatic Embryogenesis, a Means to Obtain Transgenic Plants
Scheiden and Schwann made a critical observation, that plants possess a remarkable
ability to generate free-living cells from plant tissues. Plant cell and tissue culture using
sterile technique and in vitro (within glass) conditions are key elements to obtaining
transgenic crop plants.
Somatic Embryogenesis
Totipotentcy: plant cell or cells are able to live independently, and also possess the ability
to regenerate into a whole plant under the right environmental conditions.
The first methodology of generating plants from cell culture is through the process of
somatic embryo formation, whereby somatic cells (those not involved in sexual
reproduction), produce an embryo similar to one produced by zygotic embryogenesis it is
called somatic embryogenesis.
Somatic embryos formed with root and shoot apical meristems are termed a bipolar
embryo, and germinate into whole plants (Parrott, 2000).
Organogenesis
The second methodology of regenerating single cells into whole plants is termed
organogenesis, where a meristematic cell from a root or shoot primordiums used to form
organs (e.g. shoots, leaves or roots); these recovered organs can then be cultured into whole
plants.

9. Methods of Plant Transformation
The process of introducing DNA into living cells is called transformation.
Introduced DNA is a viral DNA ,the process is called transfection.
MICROINJECTION
• DNA solution is injected directly inside the cell using capillary glass micropipettes with
the help of micromanipulators of a microinjection assembly.
• Involves the use of a micro-capillary to inject either the
target DNA directly into the nucleus or the target RNA
directly into the cytoplasm
Partical Gun / Biolistics / Macroprojectile
A device for injecting cells with genetic information
The payload is an elemental particle of a heavy metal
(tungsten,gold,silver) coated with plasmid DNA.
This technique is often simply referred to as bioballistics or
biolistics.
 Helium propellant and a multi-disk-collision delivery
mechanism is used.
 Target of a gene gun is often a callus on gel medium in a
petri dish

10. ELECTROPORATION
 Exposing cells for very brief period of electric pulses-induce
transient pores in plasma lemma.
Fiber mediated transformation
Using silicon carbide fibres of 0.6micro m in diameter,10-80
micro m length.
Chemical methods involves……….
1) Lipofection
2) PEG mediated transfer
3) Capo4 precipitation method
4) DEAE Dextran method

11. Vector mediated transfer………
 Agrobacterium mediated transformation
Transformation of plant cells by Agrobacterium tumefaciens harboring a wild-type Ti plasmid

12. Molecular Biology for Plant Transformation
Overexpression Vectors
 The importance of promoters in vector construction; as the choice of a
promoter sequence and its optimization determines the constitutive, spatial, and/or
temporal expression of transgenes
Promoters for the Expression of Transgenes
The regulation of gene expression is one of the goals in the development of transgenic
cultivars. Promoters, enhancers and cis-acting regulatory sequences are all DNA
elements, which control gene expression. Biotechnology has developed the ability to
engineer chimeric expression cassettes, by combining promoters and coding regions from
different genes.
RNA Interference
Gene silencing has resulted in the production of transgene cassettes capable of silencing
specific genes in a dominant fashion. This process is termed RNA interference, or RNAi,
and results from the production of double-stranded RNA (dsRNA) in the plant cell. The
dsRNA produced specifically prevents the production of protein from messenger RNA of
the target gene
Zinc-Finger Nucleases
Zinc-finger nuclease technology allows for constructs to be designed to target transgene insertion into a
specific locus within the plant genome , used to produce knockout mutations by disrupting the sequence
of the target gene; i.e. causing a frame-shift mutation, elimination of the start codon or important motif,
or a complete removal of the target gene.

13. Breeding with Transgenics
After obtaining a transgenic event, the genetic engineer hands the seeds over to
the plant breeder.
Lines that survive transformation and tissue culture well are typically lower yielding
than current elite lines. To make these transgenic lines marketable, plant breeders
must use breeding techniques to transfer the transgene into a high yielding elite line

14. • First, the breeder obtains an inbred line by self-pollinating the transgenic line. Each
plant cell in the transgenic inbred line now contains two copies of the gene.
• The transgenic seeds produced are planted along with seeds from an elite inbred.
• Due to a lack of hybrid vigor, both of the inbred plants that grow from these seeds will
be smaller than current hybrids.
• When the plants reach the proper stage, they are cross-pollinated.

15. • The seed from this crop, the F1 seed, is harvested. All of the offspring have one copy of
the transgene, as well as 50% elite and 50% non-elite genes.
• The F1 seed is planted near another elite inbred seed. The plants grow. Due to hybrid
vigor, the F1 plant is larger than the elite inbred, but it still contains many undesirable
genes.
• The F1 plant is mated back to the elite inbred. This process is known as backcrossing.

16. • This seed, the backcross 1 (BC1) generation is harvested. The plants that grow from
these seeds will have 75% elite genes, and half will contain the transgene.
• Again, BC1 plants are grown along with elite inbreds. Those that express the
transgene are cross-pollinated with the elite inbreds.

17. • This process is continued until the plants contain at least 98% of the elite genes and
the transgene. This takes approximately 5 to 6 generations.

18. Comparison
Classical breeding Transgenic breeding
Desired gene is
incorporated along
with many other
genes ,which then
have to be bred out
Desired gene only is
transformed from one
organism to another
Crosses limited by
species barriers
Species barriers may
be overcome

19. Transgenic Breeding for Increasing Yield
Crop Non transgenic (yield
per ha.)
Transgenic (yield
per ha.)
Improvement in
yield by %
1. Cotton 8-10 qtl. 14 qtl. 14
2. Corn
(Forage)
50-60 qtl. 70 qtl. 7
3.Soybean 20-22 qtl. 28-30 qtl. 20
(Source- CICR Technical bulletin – 22).

20. Transgenic Breeding for Insect Resistance
Crop Gene
inserted
Resistance to Vector Achievement Year
1) Cotton Cry gene
complex
Bollworm complex Agrobacterium
tumefacience
1) 1 st Bt cotton in world
by Monsanto Ltd. USA.
2) 1st Bt cotton in india -
1996 by MAHYCO -
Monsanto Ltd.
1987
1996
2) Brinjal Cry 1 Ab Fruit and shoot
borer
Agrobacterium
tumefacience
1) 1 st transgenic (Bt)
Brinjal in world ( USA)
2004
3) Cabbage Cry gene
complex
Cabbage worm
and cabbage
looper
Agrobacterium
tumefacience
1) 1 st transgenic (Bt)
Cabbage in world (USA)
2002
4)Maize Cry gene
complex
European Corn
Borer
Agrobacterium
tumefacience
1) 1st Transgenic maize in
world is Maximizer (Spain) by
Company Ciba Geigy
1995
5)Okra Cry 1 Ab Fruit and shoot
borer
Agrobacterium
tumefacience
1) 1 st Transgenic okra in
world by U.S.A.
2002

21. Crop Gene inserted Resistance
to
Vector Achievement Year
6) Potato Cry-gene complex Colorado
potato beetle
Agrobacterium
tumefacience
First transgenic potato in
world – Monsanto Ltd. U.S.A.
1995
7) Apple Cry-1-Ac Codling moth Agrobacterium
tumefacience
First transgenic Apple in world
by U.S.A.
2000
8) Soybean Cry-1-Ac Leaf eating
catterpillar
Agrobacterium
tumefacience
First transgenic Soybean in
world – Monsanto Ltd. U.S.A.
1996
9) Sugar cane Cry-1-Ab Stem borer Agrobacterium
tumefacience
First transgenic Sugar cane in
world – U.S.A. DNA plant
technology company
1997
10) Tomato Cry-1-Ac Tobacco horn
worm
Agrobacterium
tumefacience
First transgenic Tomato in
world – U.S.A by Calgene
company
1987
11)Tobacco Trypsin inhibitor
gene from
cowpea
Leaf eating
catterpillar
Agrobacterium
tumefacience
First transgenic tobacco
resistant to leaf eating
catterpillar was developed in
USA.
2001.
Source – Esseentials of Plant Breeding

22. Transgenic breeding for keeping quality
Crop Gene inserted Traits Vector Achievement
1) Tomato - a) Delay
ripening
b) Delay fruit
softening
c) Thicker skin
and Alter
pectin
content
a) Agrobacterium
tumefacience
b) Antisense RNA
technology
c) Agrobacterium
tumefacience
a) Variety :
Endless
summer by
DNA plant
technology
company
1995
b) Flavr Savr by
Calgene
company in
1994
c) By Zeneca
company
1995

23. Crop Gene inserted Traits Vector Achievement
2) Apple ACC oxidase gene Delay ripening Agrobacterium
tumefacience
In scion cultivar
ROYAL GALA is
developed
3) Banana ACC gene Delay ripening Agrobacterium
tumefacience
Cavendish 1
4) Mango Rol C gene Storage and
delay ripening
Agrobacterium
tumefacience
Golden yellow
( Source - AGROBIAS Newsletter. 2006).

24. Transgenic breeding for disease resistance
Crop Gene inserted Resistance to Vector Achievement
1) Potato PLRV-R PVX, PVY, PLRV Agrobacterium
tumefacience
First transgenic
potato in world by
– Monsanto Ltd.
U.S.A. 2000
2) Cumcumber GUS and Markar gene
NPT II
CMV, ZYMV, WMV 2 Agrobacterium
tumefacience
First resistance
variety Beit alpha
MR
3) Faba bean Chitinase gene from
Seratica marcescence
Chocolate spot
disease
Plasmid First resistance
variety in 2002
4) Banana FR gene Panama wilt Plasmid First resistance
variety isCavendish
5) Papaya PRVR gene Papaya ring spot
virus
Plasmid First resistance
variety is Sunup
and second variety
is Rainbow in 1999
Hawai.

25. Transgenic breeding for Herbicide Resistance
Crop Gene transfer resistance to Vector Achivement
1.Rice Bar chimeric gene Hygromycine Plasmid Oryza sativa cv. IR 72
2.Nilgiri (Eucalyptus) Cry 3A Glufosinate
ammonium(Liberty@6
l/ha)
Agrobacterium
tumefacience
Variety-Ecofriend
3.Sugarcane PPTR(
phospinothricine
resistant gene)
Phosphinothricine Agrobacterium
tumefacience
I st herbicide resistant
sugarcane was
developed in USA.
4.Cotton Phosphinothric acetyl
transeferase
All non selective
herbicides
Particle gun
bombardment
method
I st variety was
releasedIn year 2000.

26. Crop Gene transferred Resistance to Vector/
Transferred by
Achievement
5.Pineapple GUS (B-glucuronidase)
and Bialphos resistant
(Bar) gene
BASTA Microprojectile
bombardment
method
Var -Bialphos Ready
6.Sugarbeet Rr gene ( roundup
resistant gene)
Round up Microprojectile
bombardment
method
Variety-H7-1
Released by Monsanto
Ltd. in 2008 in USA.
7.Soybean Rr gene ( roundup
resistant gene)
Round up Microprojectile
bombardment
method
I st Roundup resistant
var. in soybean was
released in 1997 in
USA.
(Source –www.google.co.in)

27. Transgenic breeding for adding Male sterility
Crop Gene inserted Source of gene
1.Rapeseed mustard msi (male sterility iducing
gene)
Bacillus
amyloliquefacience
Source – Essentials of Plant Breeding ,
Transgenic breeding for drought resistance
1.Drought resistance in sorghum
Gene transformed mlt D gene
Vector Agrobacterium tumefacience
Developed at CRIDA, Hyderabad
Number of transgenics released 14.

28. 2. Drought resistance in Wheat
1. Crop American bread wheat
2.Genes transformed mlt D and
HVA 1.
3.Source of gene Osmotin gene complex from
Atriplex numularia
4. By Microprojectile bombardment
system
Transgenic breeding for changing morphological character
1. Crop Petunia
2. Character modified Orange flower colour
3.Gene transferred Dihydroflavanal 4 reductase
(FR) gene.
4.Source of gene Zea mays L. (Corn)
Original Petunia
Modified Petunia

29. • Increasing grain yield and fodder yield.
• Creation of disease resistant, pest resistant,
adding quality characters, inducing male sterility,
production of herbicide resistant, metal toxicity
resistant crops.
• Reduces cost of cultivation.
• Reduction in air pollution caused by use of the
pesticides.
• An ecofriendly method.
Source – Essentials of Plant Breeding
Advantages of Transgenic Breeding

30. Disadvantages of Transgenic Breeding
 High cost of seed production.
 Require high skill.
 Require high cost facilities for laboratory work.
 Screening of large number of protoplasts is essential.
 Occurance of minor pests increases.
 Production of the resistant generations of pests.
 Transfer of only monogenic character is possible at a
time.
• Unstable performance at several locations.
• Monopoly of private seed companies
Source – Essentials of Plant Breeding