ADVANCES IN VEGETABLE IMPROVEMENT FOR QUALITY AND QUANTITY

1. M.Sc Course seminar on the topic
Department of Horticulture
Banasars Hindu University
Varanasi-221005
Supervisor : S.P Singh
Saurabh Singh
I.D. NO. H-15130
M.Sc Ag final yr.

2. Introduction of Biotechnology
Term Biotechnology was coined by Karl Ereky, a
Hungarian engineer, in 1919.
According to Rai and Rai, the origin of Biotechnology
traced back to prehistoric times when farmers and
gardeners started the use of grafting and hybridization
among species with a view to bring an improvement in
existing attributes of a species or variety.

3. DEFINITION
 Controlled use of biological agents, such as micro-
organisms or cellular components, for beneficial use (
U.S. National Science Foundation).
 Bios = life , technos = tool, logos = study of
Thus, Biotechnology = study of living tools.

4. Conventional breeding
• Long time trails
• Involving high cost
• Linkage drag (Transfer of undesirable genes along with
desirable gene and in the process of transfer we may loss
some other useful genes from original genome).
• Distant Hybridization barriers

5. Cont………
• Inbreeding depression in cross pollinated plants
• Ineffective phenotypic selection
• Difficult to produce viral resistant plants in case of
clones.
• Not possible to transfer a gene from micro organisms
• In effective diagnosis of diseases

6. Why to use Biotechnology approaches ?
• Eliminates long term trails
• 100% achievement of gene transfer, no transfer of undesirable
genes, no loss of genes from original genome.
• Over coming Distant Hybridization barriers
• Development of inbreds without Inbreeding depression in
cross pollinated plants
• Effective Marker Assisted selection
• Production of viral resistant plants in case of clones.
• No species/genus barriers
• Production of genetically true to type plants

7. Non conventional breeding method
• Tissue culture
– Anther culture
– Embryo culture
– Somatic hybridization
– Somaclonal variations
– Protoplast fusion
– Micro propagation
• Genetic engineering (recombinant DNA technology)
• Markers

8. The capacity of a plant cell to regenerate into a
whole plant ( Vochting, 1878).

9. Meristem Culture
 First application of meristem culture done to obtain
virus-free plants of dahlias (Morel & Martin 1952).
 Apical Shoot and root meristem free from virus
infection .
Due to high auxin concentration.
High metabolic activity due to actively
growing meristem cells.
Absence of vascular system in meristem.

10. STERILIZATION
CULTURING IN MEDIA
(MS media)
ADDITION OF GROWTH ADDITIVES
(SUBCULTURING)
REGENERATION OF SHOOTS AND ROOTS
PROTOCOL FOR MERISTEM CULTURE

11. Crop Purpose Ref.
Onion Elimination of onion yellow
dwarf virus
Walkey et al. 1987
Shallot Elimination of onion yellow
dwarf virus
Walkey et al. 1987
Pea Elimination of pea seed
borne mosaic virus
Kartha and
Gamborg 1978
Achievements of Meristem culture

12. Anther culture
 Isolation of homozygous lines.
 Hybrid development.
 Induction of mutations .induction of genetic variability.
 Generation of exclusively male plants in asparagus.
 Hybrid sorting.
 Double haploids in genome mapping .
 Its application restricted in few spp.
Explant- pollen
XX YY
XY
(ALL MALE PLANT)
Anther may be either X ‘‘OR Y’’
Y is cultured and DH YY developed ,
its pollen used for pollinating female

13. ANTHERS COLLECTED FROM STERILIZED FLOWER BUD
STERILIZATION ( NaClO)
SQUEEZING TO RELEASE MICROSPORES BY PRESSING
REMOVAL OF ANTHER DEBRIS THROUGH FILTERATION
VIABLE AND LARGER MICROSPORE SELECTED
CENTRIFUGATION OF POLLEN SUSPENSION AT LOW FREQUENCY
SUPERNATANT DISCARDED AND PELLET OF POLLEN RESUSPENDED IN FRESH MEDIA , WASHED
OBTAINED MICROSPORE MIXED WITH APPROPRIATE CULTURE MEDIA

14. FINAL SUSPENSION PIPETTED INTO SMALL PETRI DISHES , LAYER OF LIQUID IN SUSPENSION
SHOULD BE THIN FOR PROPER AERATION
SEALING WITH PARAFILM TO AVOID DEHYDRATION
RESPONSIVE MICROSPORE FORM EITHER EMBRYO OR
CALLI
SUBSEQUENT PLANT DEVELOPMENT BY TRANSFERRING TO SUITABLE MEDIA
Continued..

15. Pollen on media
Callus formation Shoot initiation
Root initiation Whole plant
Source:
TNAU portal

16. ANTHER CULTURE
CROP ACHIEVEMENT REF.
Tomato MS purelines Schereva et al.
1990
Chilli Haihua 3 Variety Li & Jiang 1990
Homozygous resistant
line for PV Y
Selassie et al.
2006

17. EMBRYO RESCUE
 Explant – immature
embryo
 Used for overcoming post
fertilization barriers
(EMBRYO ABORTION) in
distant hybridization.

18. SOMATIC HYBRIDIZATION
The fusion of the two somatic cells of different species, genera or family are
considered as somatic hybridization (Rai &Rai 2006).

19. CYBRID Hybrid
N1
N2
N1
N1
N1 N2
N1+N2
Contains the two protoplasm
and nucleus- Allopolyploids
N1
N2
Hetero
karyon
SOMATIC HYBRIDIZATION

20. APPLICATIONS OF SOMATIC HYBRID
 Production of novel intergeneric and interspecific crosses .(eg
pomato)
 Used in disease resistant gene transfer , e.g leaf blight ,
phytopthora resistant gene transferred to Solanum tuberosum.
 used in abiotic stress resistant gene transfer e.g -
Solanum tuberosum Solanum acaule
SOMATIC HYBRID
( FROST AND DISEASE RESISTANT)

21. CONTINUED
For ameliorating quality trait-
Brassica napus Eruca sativa
Cybidization –
Cabbage protoplast ogura CMS Brocolli lines
Somatic hybrid
(with low erucic acid content )
Cold tolerant CMS Cabbage

22. Achievements
S.melongena +
S.integrifolium
Resistant to
Pseudomonas
Kameya et al.
1989
S.melongena +
S.saintwongsesi
Resistant to
Pseudomonas
Asao et al.
1994
Broccoli +
Cauliflower
Transfer of CMS-
ogura (R.sativus)
Sigareva,
Earle 1997
S.tuberosum +
S.brevidans
Resistant to PLCV,
Late blight
Helgeson et
al. 1989
L.esculentum +
S.muricatum
(pepino)
For various desirable
characters
Sokomoto,
Taguchi 1991

23. SOMACLONAL VARIATIONS (SCV)
• The generation of considerable variation, which is
heritable during tissue culture is known as SCV
(Rai & Rai 2006).
• Variations can be created for:
I. Higher yield
II. Herbicide resistance
III. Fungal toxin resistance
IV. Temperature tolerance
V. Salinity tolerance

24. Recombinant DNA technology
•The R-DNA technique involves breaking of a DNA molecule at two
desired places to isolate a specific DNA segment and then inserting
it into another DNA molecule at a desired position. The product thus
obtained is called Recombinant DNA and this technology is known
as R-DNA technology (Jakhar & Sastry 2002).
•Transgenics
• A plant in which a foreign has been transferred through
genetic engineering is called a transgenic plant and the gene
so transferred is called transgene (Singh 2001).

25. Agrobacterium tumifaciens
plasmid

26. Mechanism of Viral Resistance
• Coat proteins synthesized in plants block the receptor
proteins, which are essential for the uncoating of viral
nucleic acid.
• They interfere with the viral replication or expression
of viral genes.
• Anti sense RNA technique- which inhibits viral
protein synthesis.

27. The transgenic summer squash plant (upper left) is resistant while the conventional plants are
susceptible to single infection by CMV
(bottom left), ZYMV (bottom right) and WMV (upper right)
(Photo by M. Fuchs 2008).

28. • Bt denotes Bacillus thuringiensis, a soil borne gram
positive bacteria, whose genes produce delta endotoxins
that are toxic to insect pest. These toxins interfere with
ion uptake in alkaline insect midgut.
• They are not toxic to humans and animals because it
works in the alkaline digestive track of insects.
Bt- insect toxin

29. Engineering for Abiotic stress
resistance
• Heat shock protein encoding gene HSP17.7 is found
in carrot (Malik et al. 1989) which can be transferred
to other species for inducing thermal stress.
• Anti freezing protein gene has been transferred from
winter flounder fish to tomato and soyabean
(Hightower et al. 1999).

30. Flavr Savr
Supermarket
Ripe and Flavoured.
Traditional
The traditional
tomato must be
harvested while it
is mature green
(before they
attain the flavour)
to facilitate long
distance
transport, so that
it is not crushed
on the way to the
supermarket.
The traditional
tomato is
sprayed with
ethylene after
transport to
induce ripening.
Supermarket
Ripe but doesnot contain
original flavour
We can harvest the
tomato fruits for long
distance transport
after
attaining full flavour
in breaker stage.
Due to inhibiting the cell
wall pectin breakdown
after breaker stage it
remains firm during
transport and don’t
bruise & don’t loss the
flavour

31. Marker
Markers are the heritable characters whose inheritance
pattern can be followed at the morphological, biochemical
and molecular level.

32. MARKER ASSISTED SELECTION
 Foreground selection
 Background selection
RP
DP
GENE A
GENE aMARKER b
MARKER B
Selection for marker B
GENE AMARKER B
RECURRENT PARENT
CHROMOSOME
DP
RP
Gene A
Gene a
M-B
M-b
M-D
M-c
M-C
M-d
M-B GENE A
M-d M-c

33. Bioinformatics is a new science that combines the power of
computers, mathematical algorithms, and statistics with concepts
in the life sciences to solve biological problems. Through
bioinformatics, scientists have been able to analyze various
genomes.
Data
DNA, RNA, Protein
sequences
Molecular
structure
Bibliographic data
Expression data
Bioinformatic tools

34. How bioinformatics improve biotechnology
Basic research
Design of better plants
Harness genetic diversity
New tools to study gene function
Test, analyze and identify plants

35. Drawbacks
• High Cost involved in research (Equipment & Lab)
• The stability of transgene expression has not always
been assessed.
• Requirement of tissue specific promoters (MS,
Parthenocarpy, Ripening)
• Controlled gene expression –(E.g. Cpt)
• Ecological disturbance
• Culture contamination problems
• Pathogen, Insect biotype
• Most of them are of academic purpose only
• Superweed development

36. Future Prospects
 Status of biotechnology in India is encouraging.
 Future back on with many opportunities.
 Require large resource and development investment.
 Require allocation of fund for holistic growth of
building scientific capabilities and knowledge.
 Biotechnology is unnatural process ,hence ignore the
dynamic changes in genetic code.
 Its application restricted for novel and valuable traits
in short-rotation spp.

37. Continued..
• To obtain transformed plants from recalcitrant
spp.
• Mapping of genome.
• Increasing the no. of isolation of genes
conferring resistant to biotic and abiotic
stresses.
• Production of artificial seeds.
• Patent legislation is important to protect new
gene products.

38. Conclusion
Biotechnology Bioinformatics Improvement
in agriculture
Establishment of National Biosafety System
required to encourage the growth of domestic
biotechnologies and to ensure safe access to new
products and technologies.

39. THANK
YOU