This document discusses various applications of biotechnology in plant breeding and agriculture. It describes techniques like anther and pollen culture that are used to produce haploid plants. It explains the process of protoplast isolation and culture, and its importance in genetic transformation and hybrid production. Transgenic cotton containing Bt genes that provide resistance against bollworms is discussed. The document also outlines the production of synthetic seeds through encapsulation of somatic embryos, and the advantages this provides. Finally, it summarizes some key achievements and developments in crop biotechnology like increasing yield, improving stress resistance, nutritional quality, and reducing dependence on agrochemicals.

1. By
Mr.Pranav V. Gadkar,
Assistant Professor,
Department of Botany
BNB College, Digras
Applications of Biotechnology

2. Haploid Plant Production
 Anther Culture
 Technique of growing excised anthers on nutrient
media
 Microspores develop into callus or embryoids
 From callus plantlets are regenerated

3. Androgenesis
 In vitro regeneration of haploids plantlets from pollen
grain
 Direct Androgenesis- pollen grain behaves like
zygote & form embryoids
 Indirect androgenesis- Pollen grain develop callus
from callus haploid plantlet developes

5. Anther Culture
 Basic Principle of anther and pollen culture is
regeneration of haploid plantlets by microspore
totipotency
 Protocol
Collect the flower buds
Transfer selected flower to laminar & Surface
sterilise it

6. Remove the stamens with sterile scalpels
Excised anthers & inoculate them on MS media
Kept culture in dark
Haploid plantlets appear
Hardening of plantlets

8. Pollen Culture
 Pollen are cultured on media
Collect the flower bud
Transfer buds to laminar
Remove stamens with sterile scalpel
Excised anthers press it against wall of glass to
Squeez out pollen

9. Centrifuge the pollen suspension at 500-800 RPM
Suspend pollen pellets in liquid media
Subculture on soilid media
Young embryoids appear after 30 days

10. Advantages of anther Culture
 Unwanted growth of anther wall eliminated
 Androgenesis can be observed from single cell
 Pollen is ideal for transformation and mutagenic studies
 More haploids are regenerated
Importance of Anther and Pollen Culture-
 Mutation study
 Plant breeding programs
 Cytogenetic studies
 Haploid germplasm bank
 Secondory metabolite production
 Clones

11. Protoplast Culture
 Principle of plant protoplast culture is aseptic isolation of large
protoplast & culture them on media
Isolation of Protoplast
 Suitable material is leaf messophyll
 Removal of cell wall without damage
 Cells stabilised by mannitol
 Removal of cell wall by 2 methods
Mechanical Method-
 Breaking of cell compartment under microscope
Enzymatic Method-
 Cell wall degrading enzymes used like cellulase, pectinase,
macerozyme
 Provides large number of protoplast in short time

12. Protoplast Culture
Isolated protoplast are suspended in basal liquid media
Solidified agar of sealed petridish melted then cooled
To this medium add 1.5 ml of protoplast suspension added
Petridish inverted and incubated at 25 degree

13. Importance of Protoplast Culture
 Effects of environmental factors on osmotic
behaviour of cell
 Useful in establishing role of IAA in Cell elongation
 Biochemical and biophysical aspects of
photosynthesis
 Best studied for high efficiency of gene transfer
 Explitation of gene variability of cultured cell
 Production of somatic hybrids between distantly
related species

14. Somatic Hybridisation
Potato plant Tomato plant
Protoplast Protoplast
Heterokaryon
Growth of hybrid
colonies
Selection of Hybrid colonies and planting on
media
Hybrid callus and
plantlet
Fusogenic
chemicals

16. Types of Protoplast Fusion
 Spontaneous fusion-
Simple physical contact is sufficient between similar parental
protoplast
 Induced Fusion-
-fusion of protoplast from different sources with help of chemical
agents
a. Mechanical Fusion- In this method protoplast brought into close
contact under microscope using micromanipulator
b. Chemofusion- In this method several chemicals are used to induce
protoplast fusion like sodium nitrate , PEG, calcium ions, Polyvinyl
, alcohol called chemical fusogens
c. Electrofusion- electrical stimulation used to fused protoplast, two
glass microelectrodes are palced in contact with protoplast
suspension, subsequent application of electric current for some

17.  Importance of Protoplast Fusion
1. Used to improve plant species through hubrid
production
2. Methods for combining different genomes
3. Used to overcome sexual incompatibility barrier
4. Fusion product provide information about
compatibility or incompatibility

18. Transgenic Bt Cotton
 BT Cotton is GM Variety of cotton
 Produce insecticide
 Produced by Monsanto Company
 Bacterium Bacillus thuringiensis (Bt) produces
harmful chemicals to selective insects
 The CRY 1Ac gene coding for Bt Toxin (cry
proteins) inserted into cotton
 It Eliminates the need to use large amounts of
broad spectrum insecticides
 Cotton incorporated with cry 1Ac gene makes it
toxic to bollworms

20. Modeof action of Bt Cotton
 When insect larva attack and eat cotton the cry
protein are dissolved
 Dissolved and activated cry proteins molecules
bound to receptor proteins on epithelial cell
comprising brush border membrane of epithelial
cell of midgut.
 Ion Channels formed in cell membrane which allow
flow of potassium ions
 Regulation of potassium ion lost and results in
death of epithelial cell.
 Death of epithelial cell creates gap in brush border
membrane resulting in death of larvae

21. Mode of actionof BT Cotton

22. Synthetic / Artificial Seeds
 Living seed like structures
 Produce experimentally by encapsulating the
somatic embryos derived from plant tissue culture
in a hydrogel
 Behaves like true seeds if grown in soil
 Radenbangh et al found that sodium alginate could
be used to produce artificial seeds

24. Importance of Artificial/Syntheticseeds
 Made available within one month. Nobody has to
wait for long time.
 Production is not time dependent or season
dependent
 Useful to shorten life cycle of plant
 Used to overcome dormancy period
 Help to study the role of endosperm and seed
coat formation

25. SalientAchievements of Crop Biotechnology
Some of the recent developments in crop
biotechnology are as given below-
A. Crop Yield- One or more genes may be
transferred to a highly developed crop variety to
introduce new characters that would increases
its yield
B. Reduce Vulnerability of crops to environmental
stresses- Crop resistant to biotic and abiotic
stress have been developed. Eg. Plant gene At-
DBF2 from Arabidopsis thaliana shows
tolerance to salt, drought, heat and cold. Rice
plant resistant to RYM Virus created.

26. C. Increased Nutritional Qualities- Proteins in food
require modification to increase its nutritional
qualities. Protiens in legumes and cereals may be
transformed to provide amino acids needed by
human beings for balanced diet.
Eg. In Golden rice scientist introduced three genes
associated with biosynthesis of carotenoids.GM
rice produces beta carotene which is converted to
vitamin A.
D. Improved taste, texture or appearance of food-
Modern Biotechnology used to slow down the
process of fruit, vegetable , oil seed spoilage.
Modified fruits can ripen longer and
transported.Researchers modified tomato and

27. E. Reduced Dependence on fertilizers, pesticides and
other agrochemicals-
-Bt produces bt toxin with insecticidal qualities.
- Crops have been engineered to aquire tolerance to
broad spectrum herbicide. The herbicide tolerant
crops have potential of increasing yield .
- Transgenic crops that express tolerance to
glyphosate, glufosinate and bromoxynil have been
developed.
F. Production of Novel substances in crop plants-
- Oil seeds can be modified to produce fatty acids for
detergents
- Potatoes, tomatoes, rice, tobacco, lettuce,
safflowers have been genetically engineered to
produce insulin and vaccines.

28. G. Glyphosate resistance- One of most famous kinds
of GM crops are Roundup ready or glyphosate
resistant crops. Glyphosate kill plants by intefering
with shikimic acid pathway in plant,
H. Improved shelf life- The first genetically modified
crop approved for sale in us was Flavr savr tomato
which had longer shelf life, A gene in fruit has been
modified in such way that apple produces less
polyphenol oxidase , a chemical that manifests
browning
I.Pathogen resistance- Tobacco , Corn, rice and many
other crops have been generated that express
genes encoding for insecticidal proteins from Bt