Transgenic plants development

1. SEMINAR TOPIC
PRESENTED ON:28-06-2022
Dr. GOWDRA NAGAMMA
M.Sc.4th SEMESTER

2. CONTENTS
• INTRODUCTION
• HISTORY OF PLANTBIOTECHNOLOGY
• CONVENTIONAL PLANT BREEDING
• DIFFERENT APPROCHES OF CROP IMPROVEMENT
a.PTC
b.TRANSGENIC APPROACHES
c.CHEMICAL METHODS
d.MOLECULAR BREEDING METHOD
• ENHANCING PRODUCTIVITY THROUGH GM CROPS
• BIOTECHNOLOGY FOR IMPROVING NUTRIONAL QUALITY-TRANSGENIC
PLANTS FOR BETTER HUMAN HEALTH
• SUMMARY
• CONCLUSION
• REFERENCES

3. INTRODUCTION
Plant biotechnology has played a significant role in crop improvement and
productivity.Plant biotechnology is a tool that intensively use the biological
systems to produce quality products for human consumption,particularly in the
field of agriculture and food processing.
The word biotechnology is derived from the greek words “bios” means all
to do with living system and “technikos” means including human knowledge
and skills.
Plant biotechnology may be defined as a set of biological tools and
techniques used in plants for the development of products of commercial value.

4. Some basic techniques of tissue culture such as anther
culture,somaclonal variation, embryo culture and somatic hybridization
are being explorted to generate useful genetic variability for obtaining
improvement in commercial cultivars.
Plants are also used for molecular formingpharming for the
production of secondary metabolites, such as
food,flavours,colors,dyes,perfumes drugs and scented oils used in
aromotherapy through cell culture and hairy root culture.

5. • The history of PB can be traced back to the history of cell and tissue
culture,which had its birth with the demontration of totipotency of plant
cells by G.Heberlandt.
• Heberlandt was the first to conduct experiment designed to demonstrate
totipotency of plant cells by culturing isolated leaf cells in diluted Knop’s
nutrient solution.He failed largelly because of the poor choice of
experimental materials,inadequate nutrients and infection.
• Nevertheless,he boldly predicted that it should be possible to generate
artificial embryos(somatic embryos) from vegetative cells, which
encouraged subsequent attemts to regenarate whole plants from cultured
cells.

6. CONVENTIONAL PLANT BREEDING
• Since the beginning of agriculture, farmers have been altering the genetic
makeup of the crops they grow.
• Early farmers selected the best looking plants,seeds and saved them to grow or
plant for the next year.The selection of features such as faster growth,higher
yield,pest and disease resitance,larger seeds or sweeter fruits has dramatically
changed domesticated plant species compared to their wild relatives.
• The plant breeding came into being when man learned that crop plants could be
artificially mated or cross pollinated to be able to improve the characters of the
plant.
• Desirable characters from different parent plants could be combined in the
offsprings.This conventional plant breeding used to develop new varieties of
crops for 100s of years.

8. MUTATIONAL BREEDING
• The recognisation of desirable traits and incorporating then into
future generation is very important in plant breeding.A few of these
traits occationally arise spontaneously through a process called
mutation but the natutral rate of mutation is very slow and reliable
to produce plants that breeders would like to see.
• In the late 1920s, researchers discovered that they could greatly,
increase the no. of these variations or mutations by exposing plants
to x-ray and mutation inducing chemicals.
• In 73 years of mutation breeding(1939-2013) a total of 3218
varieties obtained through MB have been registerd in the IAEA data
base.
• Staple crops such as rice has registered 824, barley 312,wheat 274,
maize 96,tomato 20,potato 16,sugarcane 13 varieties.

10. DIFFERENT APPROACHES OF CROP
IMPROVEMENT USING PB TOOLS.
1. PLANT TISSUE CULTURE 2.TRANSGENICAPPROACHES
a.Micropropagation a.Vector mediate tranformation
b.Anther culture b.Direct or physical transfermethod
c.Embryo culture c.Chemical method
d.Somaclonal variaion 3.MOLECULAR BREEDING METHOD
e.Somatic hybridization a.Marker assisted selection

11. 1.PLANT TISSUE CULTURE(PTC)
PTC broadly reffers to the in vitro cultivation of plants,seeds and plant parts on
nutrient media under closely controlled and aseptic conditions.
The totipotency of plant cells,which confers ability to differetiate in to shoots
and roots, has greatly contributed to progress in this area.
PTC is of a great value,particularly in speeding up conventional breeding and
propagation procedure.
It includes several specialised areas such as induction of callus and plant
regeneration,micropropagation,somaclonal variation,somatic
hybridization,anther culture,embryo culture and protoplast fusion etc..,
PTC method hold significant promise for creation,conservation and utilization
of genetic variability for improvement of a wide variety of crop plants.

12. MICROPROPAGATION
The term propagation as applied to plants means the generation (reproduction)
of plants by asexual means.
MP of plants is one of the best and most successful examples of commercial
application of PTC technology.It entails in vitro propagation of plants from very
small plant parts(0.2-10mm)in the lab following by their establishment in soil
under green house condition.
It is also a PTC method developed for the production of disease free high
quality planting material and for rapid production of many uniform plants.
Actively dividing young cells(meristems)are place in a special medium and
treated with plant hormones to produce many similar sister plantlets.
There are 4 stages in MP.
Methods in MP;1.Shoot tip culture 2.culture of nodes.

14. ADVANTAGES OF MP
True -to type plants produced i.e., identical to donar.
Selected plant species can be multiplied any where in the world.
Production of disease free plants.
Micropropagated,field grown plants give higher yield and exhibit better
quality.
Regenerants helps to minimize the risk of losing any genotype during
hardening and transfer to soil.
Through MP it is now possible to provide clean and uniform planting
materials in plantations-oil palm,pine,banana,date etc..,

15. SIGNIFICANCE
• Production of high quality,disease free super elite planting
material for further seed production.
• Mass production of ornamental plants.
• Rejuvenation of old varieties,propagated crops for improving
their yield and quality.
• With the PTC endangered and threatened species can be
successfully grown and conserved.

16. ANTHER CULTURE
• It is one of the best technique to improve crops by collecting anthers under
aseptic conditions from immature flowers.
• Such microspores developed in the cultured anthers are generated in to callus
or embryoids which further results in plantlets.
• The pollen within an anther contains halfdose of genome(haploid)which
spontaneously double during culture.In some species doubling of the genome
takes place by treating with colchicin.
• The doubling of the genome will allow the expression of recessive traits
which were suppressed,masked or undetected in routine plant breeding.
• There are two methods for anther culture that include 1.Direct culture
2.Indirect culture.
• This technology has been employed in the successful development of
doubled-haploid lines of rice,wheat,sorghum,barley and other field crops.

17. • Rice varieties developed through A C were released by the National seed
industry council of the Philippines since 1995.
• The first A C derived salt tolerant variety PSBRc50(bicol) was developed by
IRRI and released in 1995.
• The Philippines rice research institute developed 8salt tolerant and 2 rain fed
varieties.
APPLICATIONS:
• It can be used to improve crops by producing haploid plants.
• Improve crops of cereals,vegetables,seasonal crops like watermelon,cabbage
etc..,
• This technique could be used for developing hapliod cultures for the purpose of
developing various horticulture plants.

19. CULTURE
• In this E.C tiny embryos are excised from immature ovules or seeds and are
cultured in vitro under aseptic condition.
ISOLATION OF EMBRYO:
• The fertilized egg cells are usually excised after 1-2 weeks following
fertilization and are cultured on NA untill well establishment, then they are
transplanted into small pots and kept in a well lighted glass house for 2 weeks.
Then transplanted to larger pots.
• One common term used in E.C is “embryo rescue”,that is invitro technique
promote development a viable plant from an immature or weak embryo.
• Factors involved in the E.C:1.Media 2.Temperature and light.

21. SOMATIC HYBRIDIZATION
• It is also called somatic fusion or protoplast fusion.Somatic
hybridization is actually genetic modification for bringing the
characteristics of two distinct or same species in to a single cell or
organism in vitro by fusing the two protoplasts.
• S.H was first introduced in Nicotiana glauea. It has also been used for
the production of large number of plants mainly in Solanaceae family.
• Somatic hybrids can be produced by using two distinct or same
species and has been used for induction of resistance in potato against
virus.

23. SIGNIFICANCE
• S.H helps in forming a wide variety of recombinants among plasma
genes of different species as well as plasma genes and chloroplast
genes.
• It also helps to form hybrid cells exhibiting chloroplast genome of
one species and mitochondrial genome of another species, which is
not possible by ordinary means of hybridization of two plant species.
• These different levels of fusion and recombination help in
production of new species which have all the qualities of parent
organism or even better.

24. GENETIC ENGINEERING-TRANSGENIC APPROACH
• G.E works by physical removal of desired gene from one organism
and introducing it into another,which gives new hereditary traits to
the recipient organism encoded by that gene.
• The discoveries of 1972(first r-DNA molecules generated) and
1973(joining of DNA fragments to plasmids) started new era of
GMO’s.
• The objective of the GE is to produce high quality and safer crops for
human beings and animals.

25. METHODS IN G.E
1. VECTOR MEDIATED TRANSFER OF DNA
A. Viruses as gene vectors.
• Transfer through appropriate molecular vector of genetic information
from one organism to the other is refered to as vector mediated transfer
or transduction.
• This phenomenon was discovered in bacteria where in the DNA of an
attacking bacterial viruses( bacteriophages) gets integrated into the host
cell. The virus remains in prophase(dormant stage)and replicate along
with the host genome.
• The virus inadvently serve as carrier for the transfer of genetic
information from one bacteria to another.

26. • In addition to bacteriophage some molecular vectors of plant origin
have also been discovered these include Cauliflower mosaic
virus(CaMV) and potato leaf roll virus.
• Plant virus with genomes of DNA is few,the two main groups being
Caulimo viruses with 12 members and gemini virus with 50
members.
• Historically CaMV is the DNA virus that has received most attention
as potential gene transfer.

27. B.Agrobacterium mediated
• Another great possibility of DNA transfer into plants lies inthe Agrobacterium
infection(utilizing its plasmid as vector)
• Agrobacterium tumefaciens is a soil borne,gram negative organism,causing plant
disease “crown gall”.
• The cells isolated from crown gall grown invitro without any external source of
hormones shows tumerous or neoplastic behaviour even in the complete absence
of bacteria.
• During infection with A.tumefaciens, transfer part of a large plasmid(Ti) tumour
inducing plasmid in the nuclear genome of the infected cells.
• Super binary vectors were designed which play a significant role in the
transformation of the monocots. eg.pBIN19.pPZP,pCAMBIA vector.

29. STEPS IN AGROBACTERIUM MEDIATED PLANT
TRANSFORMATION

30. DIRECT OR PHYSICAL GENE TRANSFER
• Vector mediated gene transfer technique are restricted to certain plant
species and cell types.To overcome such limitations,physical methods
of gene transfer have been developed.
• These are based on the transformation of protoplast or intact cells
from which new plants regenerate. The most promising methods are,
1.Macro injection
2.Micro injection
3.Biolistics
4.Electroporation.

31. MICROINJECTION METHOD
ELECTROPORATER
GENE GUN METHOD

32. CHEMICAL METHOD
• Direct DNA uptake by protoplasts can be stimulated by chemicals like
PEG.
• PEG is also used to stimulate the uptake of liposomes and to improve
the efficiency of electroporation.
• PEG at high concentration(15-25%)will ppt ionic macromolecules
such as DNA and stimulate their uptake by endocytosis without any
gross damage to protoplast.
• PEG was used in combination with pure Ti plasmid.

33. MOLECULAR BREEDING METHOD
1. Marker assisted selection
• Currently molecular marker assisted breeding,an agricultural
biotechnology tool is already a routien step in breeding of most
crops,where the gene and the marker for a specific trait are known.
• These markers are short sequence of nucleic acid which makes up a
segment of DNA.
• The markers are located near the DNA sequence of the desired gene.
• Since the marker and gene are close together on the same
chromosome they tend to stay together as each generation of plant is
produced called genetic linkage.
• This linkage helps the scientists to predict whether the desired gene is
present in the plant or not.

35. ENHANCING PRODUCTIVITY THROUGH
GENETICALLY MODIFIED CROPS
1. Protecting plants from diseases.
• A major emphasis of transgenic crop development is directed to
developing host resistance to diseases.During 2001-03,plant pathogens
caused 12.5% loss in production of 6 major crop
species(wheat,rice,maize,potato,soybean,cotton) worldwide.
• In spite of the fact that use of chemical control measures has reduced
potential yield loss by 32% chemical control of diseases has inherent
environmental problems, thus the development of host resistence is a
priority for sustainable environment friendly crop protection.

36. 2.Resistance to viral pathogens
• Viral pathogens cause substantial crop loss in
beans,potato,tomato,papaya, vegetables and fruit crops.
• Encapsulation of viral genome, silencing of viral genes by antisence
RNA are the strategies to resistance against such viral diseases.
• Expression of viral coat protien coding genes in the host plant
allows the encapsulation of the invading viral DNA by CPs within
host cell,thereby preventing expression of the viral genes in host.
• The strategy was first developed by engineering CP genes of TMV
into Tobacco through which the transgenic plants delayed TMV
symptoms development.
• CPMR is widely used to develop transgenic crops resistant to
several viral diseases.

37. • Papaya ring spot virus,cucumber mosaic virus,potato virus
X&Y,wheat streak mosaic virus are the important viruses cause
severe crop losses.
• CPMR has been commercially very successful:in 1995 Asgrow
company obtained permission of commercial cultivation of squash
transgenic “freedom II” having resistance to Zucchini yellow mosic
virus and watermelon mosaic virus.

38. 3. Resistance to bacterial pathogens
• Bacteria cause a no.of serious disease in crop plants such as bacterial blight of
rice,wilt disease of vegetables,fire blight of apple,canker disease of citrus.
• The major strategies for resistance to bacterial pathogens in crop plants include
deployment of R-genes,production of antimicrobial peptides,introduction of
genes for bcterial toxins tolerance and biosynthesis of other difence related
proteins.
• Several R-genes like Ppg1 of barley,Rps1-k from soy bean,Hm 1 of maize are
have been engineered in the same species.
• In rice,resistance to bacterial blight has been obtained by transgenic expression of
endogenous Xa21gene and other Xa genes.
• Under the International program on rice biotechnology,Xa21 gene has been
integrated in to elite indica and japonica varieties &the progeny lines are used to
develop resistant varieties in different countries.

39. • Several antimicrobial peptides like attacin,magainin,defensin and
cecropin have been transformed in fruits,vegetables and forest tree
species including potato,tomato,tobacco,grapes,orange,apple for
resistance to bacterial &fungal pathogens.
• Expression of attacin,cecropin and their analogs in apple provided
resistance to fire blight disease.
• Where as in sweet orange attacin effectively prohibited citrus
canker.A transgenic tomato line containing synthetic magainin
gene exhibited considerable resistance to Psuedomonas syringae.
• Lysozyme,another antimicrobial peptide,degrades the murein
layer of peptidoglycon in bacterial cell wall,thus causing lysis.

40. 4.Resistance to fungal pathogens
• Diseases like late blight of potato,head blight of cereals,sheath
blight and blast of rice,leaf blight in maize and powdery mildew
of grapes are some of the major fungal diseases.
• Strategies for resistance include degradation of the fungal
cellwall,production of antifungal toxins.
• A potent strategy for fungal disease resistance is to synthesise
genes that can degrade fungal cell wall.
• Chitinase and B-1,3-Glucanase are cell wall degrading
enzymes,that can specifically disrupt fungal mycelium by
degrading chitin and glucan respectively.

42. 5.Insect resistance
• Insects are the second most serious threat to crop cultivation after
weeds,causing about 18%crop loss world wide.Over a period of
1964-2003,production loss has remained almost constant in
maize(9.6%-12.5%),wheat(5-9.3%) and cotton (11%-12.3%).
• Insect protection is almost entirely dependent upon chemical
control,without which potential crop loss would have risen to
24%of the total production.
• Use of insect resistance transgenic crops has reduced the use of
chemical pesticide by 37%.

43. Bt toxin engineering
• Bacillus thuringiensis(Bt),a gram positive,rod shaped,sporulating soil
bacterium,is a well known insect pathogen.
• The bacterium produces a wide variety of insecticidal toxin protiens,which
make it an efficient biological control agent.
• It produces an insoluble crystalline(cry)toxin protein known as delta-
endotoxin,which binds to receptor on the epithelial membrane of insect
midgut,causing disruption of osmotic balance and insect cell lysis.
• Bt-toxin is more specific for lepidopteran and coleopteran pests,but some cry
genes like cry2,cry3A,cry4,cry4Aa and cry11Aa are active against hemipteran
pests.
• Potato was the first commercialised transgenic Bt-crop which expresses cry3A
protein was branded as “New leaf” that provide resistance to colorado potato
beetle.

44. MECHANISMS OF Bt TOXIN

45. 6.Herbicide tolerance
• In 1994,HT tobacco was approved for cultivation,while in 1996 HT in
soybean,cotton and maize was approved.
Glyphosate tolerance:
• Soybean,maize,cotton and other crops were modified to be resistant to broad
spectrum herbicide ‘glyphosate’(roundup) thus the prefix “Roundup-ready” is
used for these HTcrops.
• Glyphosate is a nonselective herbicide which inhibits the synthesis of aromatic
amino acids from Shikimate-3-phosphate by inactivating the enzyme 5-
enolpyruvylshikimate-3-phosphate(EPSP) synthase through competition
inhibition.
• The herbicide compete with one of the substrate PEP and stops enzyme activity
by stable-EPSP-glyphosate complex and also affect the phenylpropanoid and
flavonoid pathway,killing the plant effectively.
• Transgenic crops resistance to glyphosate carry a glyphosate insensitive EPSP
synthase,coded by aroA gene from Salmonella typhimurium maintaining
normal activity in transgenic plants.

47. 7.Tolerance to abiotic stress
• In bacteria,trehalose is produced by the action of trehalose phosphate synthase
which produces trehalose phosphate and trehalose phosphate phosphatase-which
degrades the trehalose-6-phophate to trehalose.
• When these 2 enzymes are expressed in transgenic plants,the plants have larger
leaves,altered stem growth and improved response to stress.
• Over expression of glutamate dehydrogenase also improves plant growth and
stress tolerance.
• Plants have been transformed with genes encoding the alpha and beta subunits of
the chloroplast-located GDH from Chlorella sorokiniana.
• Plants with ability to produce more citric acid in roots will have a aluminium
tolerance in acid soils.
• A salt tolerant gene from mangroove(Avicennia marina) has been cloned and can
be transferred in to other crop plants.The gutD gene from E.coli can also provide
salt tolerance.

48. BIOTECHNOLOGY FOR IMPROVING NUTRITIONAL
QUALITY
1. GOLDEN RICE
• Rice being staple crop for half of the global population is the major focus for
the improvement in nutritional quality through genetic transformation.
• “Golden rice” is the rice programm which is enriched with beta-
carotene(provitamin A).
• Development of golden rice is to eliminate vit A deficiency in the rice
consuming population.
• Ingo Potrykus and Peter Beyer are credited for the engineering of phytoene
synthase gene(PSY)and lycopene beta-cyclase gene from daffodil and
phytoene desaturase gene(crt1)from Erwinia uredovora in rice grain for the
biosynthesis of Vitamin A.
• The concentration of provit A in rice grain was further enhanced by using
codan-optimized crt1 gene and PSY gene from maize upto 37microgram/g of
rice grain.

50. 2.BIOFORTIFICATION OF IRON IN RICE
• About 30%of the human population are anemic,half of which is due to iron
deficiency.
• It is the most severe mineral nutrition deficiency causing about 0.8million
deaths annually.
• Biofortification of rice with iron is a desired complementary solution to combat
mineral deficiency.
• The most widly used approach for increasing iron concentration in rice is by
expressing ferritin gene.Ferritin is an iron storage protein that can bind with
many iron atoms,which is readily absorbed by human body after intake.
• Soybean ferritin gene Soyfe H1was first transfered by
Goto,Yoshihara,Shigemoto,Toki and Takaiwa(1999) with a rice endosperm
specific promoter,which increase iron concentration in rice endosperm by
twofold.
• Nicotinamine is a mineral transporter molecule which helps in translocation of
iron and other minerals in plant body. Adeficiency in nicotinamine results in
chlorosis,a phenotype similar to iron deficiency.Expression of gene NAS from
barley under constitutive promoter result in threefold increase in iron conc.in
shoots.

51. 3.MODIFICATION OF EDIBLE OIL QUALITY
• Increase in oleic acid in oil provides benefit by reducing blood low density
lipoprotein(LDL)cholesterol.
• Conversion of oleic acid to linoleic acid is catalyzed by delta12 desaturase
encoded by FAD2-1 gene.
• In 1998,transgenic high oleic acid soybean line was commercialised by DuPont
company by silencing of FAD2-1 gene.
• Inactivation of delta12 desaturase also led to increase in the oleic acid content ut
to 89% in Brassica seed oil.
• In peanut,a transgenic cultivar SunOleic 95R was developed by Gorbet and
Knauft(1997)that contained 30% higher oleic acid than non transgenic control
plants.
• Linoleic acid is an omega-3 fatty acid pevents heart-related diseases. Over
expression of FAD3 gene in soybean resulted in 40% increase in linoleic acid
content.

52. SUMMARY
Biotechnology in agriculture has diversified applications,multiple
technologies approaches and a large reportoire of products.Plant
biotechnology is play a vital role in the improvement of crop plants
generally.This is because of its ability to overcome the shortcomings of
other conventional practices of crop improvement.
Plant biotechnology deals with cell and tissue culture,genetic
transformation, gene cloning,DNA markers and other molecular
approaches.

53. CONCLUSION
Biotechnology is a rapidly developing area of science that had
brought new ideas and tools as solution to the obvious and
unmanageable problems of food crops improvement in terms of
quality and quantity to meet out the current consumer need in all
countries of the world.

54. REFERENCES
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no.CJAST.46931
• Pratik satya ,Debabrata Sarkar(2018).Biotechnology for Sustainable
Agriculture,Chapter4 Plant Biotechnology And Crop Improvement, Elsevier
Publication.
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improvement:prospects and constraints.Plant science 163(2002)381-
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• Bello and Kadams(2003).Review:The role of biotechnology in crop
improvement. Nig.J.Biotechn.14(1)1-15.
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