1. The document discusses biotechnology applications for horticultural crops including genetically modified organisms. It describes techniques like genetic engineering, tissue culture, embryo culture and haploid breeding that can be used to develop new crop varieties. 2. Examples of traits that can be improved through biotechnology include increased yield, pest and disease resistance, and improved postharvest qualities. The document lists some GM crops that have been approved or are undergoing field tests. 3. The challenges of tropical fruit export and postharvest storage are discussed. Controlling ethylene production and perception genes could help extend shelf life of fruits like mango to increase export potential. Precision genome editing techniques may allow targeting of genes with more specificity.

1. Course Code : HRT 552
Course Title : BIOTECHNOLOGY OF
HORTICULTURAL CROPS

2. Lecture 1: Introduction to basic
biotechnology
(Importance, prospects, scope and limitations of
bio-technology in horticulture)

3. we need better and more efficient ways to
produce food
With increasing demand for food and
limiting resources...
one option is through
Biotechnology

4. Bio - life
Technology
- any technique or
procedure to develop
new products

5. Biotechnology
- any technique that uses whole or part
of a living thing to make new products,
improve or develop plants, animals
and other organisms for specific use

6. Crops biotechnology
3. Varieties resistant to pests and diseases
2. High yielding crops
4. Improved postharvest qualities
1. Tissue cultured planting materials
5. Improved food quality and food processing
5. Improved quality traits

7. Fruits – quality is indicated by sweetness,
seedlessness or soft seeded nature, freedom from
fibre, typical flavour etc
Vegetables – vitamins and mineral contents
indicate quality
Leguminous vegetables – proteins and aminoacid
Tuber crops – starch
Spices and condiments – oleoresin and essential oil
content
Aromatic and medicinal plants – essential oil and
alkaloid content

8. Genetic engineering
Technique that transfers gene(s)
of interest to develop and
improve plants, animals and
other organisms

9. - basic physical and functional
units of heredity which carries
information for the expression of
a particular trait
Gene

10. Conventional
Breeding
Wild
Relative
Crop
Plant
Genetic
Engineering
Wild
Relative
Crop
Plant

11. • limited to exchanges between
the same or very closely
related species
• little or no guarantee of
obtaining any particular gene
combination from the millions
of crosses generated
• undesirable genes can be
transferred along with
desirable genes
• take a long time to achieve
desired results
Conventional Breeding Genetic Engineering
• Allows the direct transfer of
one or just a few genes,
between either closely or
distantly related organisms
• Crop improvement can be
achieved in a shorter time
compared to conventional
breeding

12. Products developed through genetic engineering
Genetically Modified
Organisms (GMOs) = Transgenics

13. Organic agriculture and GMO’s
- GMO is not a part of Organic farming
GM crops contribute to a reduction in fuel use due to less-
frequent herbicide or insecticide applications and a reduction in
the energy use in soil cultivation. Reduced soil cultivation is
associated with herbicide tolerant crops.

14. Greenhouse
• Papaya with delayed ripening trait
• Papaya resistant to ringspot virus
Laboratory
• Mango with delayed ripening trait
• Rice resistant to tungro virus
• Vitamin A-enriched rice
• Banana resistant to bunchy top
disease
• Coconut with higher amount of
MCTs
• Sweet potato resistant to feathery
mottle virus

15. Rice help to overcome Vitamin-A
deficiency among kids

17. Genetic Engineering
 Engineering the genes
 Modifying the genes
 Insert desirable gene (Bt gene/
CryAc1)
 Insert gene of interest

18. Gene transfer to host require Vector
Commonly used vector- Plasmid (small, circular, double
stranded, self replicating extra chromosomal material of bacteria)
First recombinant DNA was constructed- Stanley Cohen &
Herbert Boyer (1972) by linking gene encoding for antibiotic
resistance with plasmid of Salmonella typhimurium
Isolation of desirable gene (antibiotic resistant)- cutting out
piece of DNA from a plasmid responsible of antibiotic resistance
which involve ‘molecular scissors’- restriction enzymes
Desirable gene/ alien DNA – linked with plasmid (vector) to
transfer into host organism
Linking of DNA involves DNA ligase- acts on cut DNA molecules
& join their ends- new combination circular autonomously
replicating DNA created in vitro- Recombinant DNA
Some basic concepts

24. Restriction enzymes belongs to nuclease class of
enzymes. Its of two types:
1. Restriction exonuclease – removes nucleotides
from the end of DNA
2. Restriction endonuclease – cut at specific
positions within DNA

25. TISSUE CULTURE

26. High Cytokinin – Less Auxin : Cell division
High Auxin – Less Cytokinin : Rooting

27. Tissue Culture (in aseptic condition)
Step 1: Growing media preparation
• Food (Murashige & Skoog / AGAR / Sucrose / Manitol / Sorbitol)
• Hormone (Cytokinin & Auxin )
• Antibiotics
• Vitamins (Vitamin B, Thiamine, Riboflavin, Folic Acid)
Step 2: Explant (Any living part of the plant)
Callus culture
Totipotency
Step 3: Cell division
Step 4: De-differentiation
Step 5: Redifferentiation
Step 6: Morphogenesis - Organogenesis - Embryogenesis
Step 7: Hardening or Acclimatization

28. USES
Rapid
True to the type
Eliminate virus (Meristem)
Production of secondary metabolites
Suspension culture
Regeneration of tissues
Organ culture– meristem, embryo, anther, ovule culture
Embryo rescue
Save endangered species
Used for those species where sexual reproduction is
tough

29. Haploid Breeding / Double Haploid
Haploid cells are produced from pollen or egg cells or from other
cells of the gametophyte
By induced or spontaneous chromosome doubling, a doubled
haploid cell is produced, which can be grown into a doubled
haploid plant.
A doubled haploid (DH) is a genotype formed when haploid cells
undergo chromosome doubling. Artificial production of doubled
haploids is important in plant breeding.
If the original plant was diploid, the haploid cells are monoploid,
and the term doubled monoploid may be used for the doubled
haploids.

30. Advantages
The ability to produce homozygous lines after a single round
recombination saves a lot of time for the plant breeders.
Studies conclude that random DH’s are comparable to the selected
lines in pedigree inbreeding.
The other advantages include development of large number of
homozygous lines, efficient genetic analysis and development of
markers for useful traits in much less time.
More specific benefits include the possibility of seed propagation as an
alternative to vegetative multiplication in ornamentals, and in species
such as trees in which long life cycles and inbreeding depression
preclude traditional breeding methods, doubled haploidy provides new
alternatives.

31. Embryo culture
Embryo culture is the culture of isolated
immature or mature embryos.
Zygotic or seed embryos are often used
advantageously as explants in plant tissue
culture, for example, to initiate callus cultures.
This embryo develops properly when
nourishing tissue; endosperm was present in
the seed during the development.

32. Use of Embryo culture
• Breeding of incompatible interspecific and
intergeneric species
• Overcoming seed dormancy
• Determination of seed viability
• Recovery of maternal haploids that develop as a
result of chromosome elimination following
interspecific hybridization
• Used in studies on the physiology of seed
germination and development

33. Protoplasm fusion
Protoplast fusion has opened up a novel
approach to raising new hybrids.
This technique of hybrid production through the
fusion of body cells, bypassing sex altogether, is
called somatic hybridization.
During enzymatic degradation of cell walls some
of the adjacent protoplasts fuse together
forming homokaryons (also referred to as
homokaryocytes, each with two to several
nuclei)

34. Somatic hybrids and cybrids
Fusion products with the nucleus of one
parent and extra-nuclear genome/s of
the other parent are referred to as
cybrid
The process to obtain cells or plants
with such genetic combination/s is
called cybridization.

35. In vitro mutation
When mutation is produced in a
segment of cloned DNA.
The DNA is then inserted into a cell or
organism, and the effects of the
mutation are studied.
Mutations are useful to geneticists in
enabling them to investigate the
components of any biological process.

36. Synthetic seeds/Synseeds
A synthetic seed is defined as an
artificially encapsulated somatic
embryo (protocorm-like bodies in case
of orchids) or meristematic tissue (shoot
tips, nodal segments, corm, and bulb)
that portrays the role of a seed and has
the proficiency of germinating into
complete plantlets.

37. Cryopreservation
Preservation at extreme low temperature.
The process of cooling and storing cells, tissues,
or organs at very low temperatures to maintain
their viability.
For example, the technology of cooling and storing
cells at a temperature below the freezing point (‘-196'
C) permits high rates of survivability of the cells upon
thawing.
• Liquid Nitrogen
• Liquid CO2

38. International Symposium Feb15-17, Rome
Biotechnology can help
Making available of cultivars with better
Storage & shelf-life
Harvest window
Classical Breeding
+ Molecular markers
New Breeding
Technologies
Decrease post harvest loss: Pre-harvest measures

40. International Symposium Feb15-17, Rome
Experiences Tomato, apple......
Some specific genes involved in
1. Production and perception of ethylene in fruit
2. Production/ activity cell-wall degrading enzymes
ACS
ACO
Ethylene
ETR
Effects
ACO-Gene
2 genes for
production
1 gene for
perception
- Other functions maintained !
- On-off switches
On
/
Off
On
/
Off
One for fruit maturation
- On-switch corrupted
- No ethylene production
- Deletion of 2 nucleotides

41. International Symposium Feb15-17, Rome
Prospects Tropical crops: Mango?
Harvested fruit: Limited storability & shelf-life
~96% production for home-market, ~4% export
 Storability & shelf improved:
● Harvesting unripe fruit
● Forced ripening close to selling:
● Ethylene + Temperature treatment
● Less tasty
http://www.fruitnet.com/americafruit/article/164144/first-aussie-mangoes-shipped-to-the-us
http://www.dawn.com/news/1182818 http://www.skymetweather.com/content/agriculture-and-economy/mango-exports-from-india-
face-a-hard-time/
http://www.ctvnews.ca/business/why-the-eu-alphonso-mango-ban-s-sweet-news-in-india-1.1809895

42. International Symposium Feb15-17, Rome
GM-fruit: Current status
Approved
 Apple No flesh browning
 Eggplant Insect resistance
 Plum Virus resistance
 Melon Storability, shelf-life
 Papaya Virus resistance
 Tomato Storability, shelf-life,
Field tests
 Banana Black Sigatoka fungus
 Orange Citrus greening

43. International Symposium Feb15-17, Rome
Storability & shelf life: Current GMO’s
Derived from two approaches:
1. Insertion foreign gene
 Redirecting pathways
 New products
 Additional antibiotic gene added to trace GMO-cells
2. Silencing:
 Blocking genes = pathways
 No new proteins produced
 Methods not specific: similar genes blocked too
 Additional gene added to trace GMO-cells

44. International Symposium Feb15-17, Rome
Storability & shelf life: Innovation
Precision Biotechnology
 Editing : Removal of a tiny DNA segment
 Promotor : Switch off !
Changes only in
 specific tissue(s)
 Specific development stage(s)
 e.g. only Mature Fruit

45. GEAC
Genetic Engineering Approval Committee

46. Some achievements of biotechnology in
horticultural crops
To be discussed in upcoming classes

47. Thank you 