This document discusses various methods for germline transformation in plants. It begins with introducing germline transformation as the process of altering an organism's genetic makeup by inserting new DNA into its genome, usually using vectors like plasmids. It then describes several methods for accomplishing plant transformation, including the pollen tube pathway method, Agrobacterium-mediated method, electrofusion, floral dip method, and biolistic method. The document provides details on the history and protocol of the pollen tube pathway method for plant transformation.

1. GERMLINE
TRANSFORMATION
Presented by:-
Eivernpreet kaur
B.Sc (botany)5th semester
180606006

2. CONTENTS
• Introduction
• History
• Plant transformation methods
❑ Pollen tube pathway method
❑ Agrobacterium mediated method
❑ Electrofusion
❑ Floral dip method
❑ Bolistic method
• Comparison
• Multiple choice questions

3. INTRODUCTION
GERMLINE
• Germline is the
population of multicellular
organism’s cells that pass
on their genetic material
to the progeny.
• Egg and sperm cells are
called germ cells,in
contrast to the other cells
of the body that are called
as somatic cells.
GERMLINE
TRANSFORMATION
• It is the process by which
genetic makeup of an
organism is altered by an
insertion of new gene(or
exogenous DNA)into its
genome.
• This is usually done using
vectors such as plasmids.

4. • Transgenic plant- It is a plant with a foreign gene (or
genes)from another plant or animal that is incorporated
into its chromosome.
• The aim of producing transgenic plants is to:-
a) Improve crop yields.
b) Improvement of varietal trait.
c)Give cultivated plants more protection against their
pests,parasites and harsh weather conditions.
▪ Several methods to accomplish plant transformation
have been devised.In all these methods,single cells
are transformed and thereafter regenerated into
complete, fertile plants by tissue culture procedures.
▪ Some of the transformation procedures can only be
applied to protoplasts.

5. • But these conventional methods of breeding require 6 to
8 generations to produce a new variety.
• Over the last decade,significant progress has been made
in the development of new and efficient transformation
methods in plants.
• Now-a-days,a new variety could be achieved in only 3 to
4 generations by introducing DNA(Deoxyribonucleic acid)
fragments.
• Despite a variety of available DNA delievery methods
such as Floral dip method,Bolistic method,Agrobacterium
tumefaciens method,Agrobacterium and Bolistic-
mediated transformation remain the two predominantly
employed approaches.

6. • Transgenes are integrated at random positions in the
plant genome,but procedures for targeted integration
become more and more efficient.

7. HISTORY
• During the experiment done by Pandey(1975) in
Nicotiana alata and Nicotiana forgetiana to overcome
self-incompatibility by “mentor pollen” technique,he
observed that pollination with highly
irradiated(100Krad)pollen can cause certain genes
transfer from pollen to the egg.
• Feasibility of this approach to transformation in higher
plants was confirmed by group of British scientists( Jinks
et al.,1981,Davies,1981).
• Pandey(1978) demonstrate intra- and interspecific
transfer of self incompatibility and flower colour alleles
from paternal genome in genus Nicotiana.
• Partial transfer of genome has been achieved in
Nicotiana,Hordeum and Triticum using this technique.

9. ✓ The technique of introduction of chemically extracted
DNA into free cells as used in prokaryotes has many
limitations when used in higher plants:-
• Expensive
• Protection
✓ Due to all these limitations,gametic transformation is
most safe,as in this process natural delievery of DNA
fragments occur via pollen tube to the egg.

10. PLANT TRANSFORMATION METHODS
DIRECT INDIRECT
Physical Chemical Biological In planta
*Microinjection * PEG * A.tumefaciens *Meristem
*Bolistics- * DEAE-dextran * A.rhizogens transformation
gene gun method * Calcium *Virus * Floral dip method
*Electroporation phosphate mediated * Pollen
*Microinjection * Proteins transformation
*Laser mediated *Artificial lipids
*Silica/carbon fibres
*SAT

11. ❑POLLEN TUBE PATHWAY METHOD
✓ First reported by Duan and Chen(1985).
✓ Its efficiency in transformation of rice was re-examined by
Luo and Wu(1989).Also give molecular evidence to confirm
the integration of DNA into genome of transformed plants.
✓ In this method,the upper portion of recently pollinated pistil
gets cut and a drop of DNA solution was applied to the cut
end of the style.
✓ The DNA presumably reaches the ovule by flowing down
the pollen tube and interacts with the genome of the
egg,leading to formation of transformed seed.
✓ This method was also used for other species as in
wheat,watermelon,soyabean,Petunia hybrida.

12. Protocol of Luo and Wu experiment in
rice plant
Rice plants grown in greenhouse at 26C and 55-60%humidity
After1-2hrs,2/3rd of florets removed by pair of scissors
A 2-3ml drop of DNA solution(50mg/ml in TE buffer)placed at
cut end of style using a capillary tube
After 40 days of treatment,seeds are harvested and
incubate at 37C for 4 days and stored at 4C
Seeds germinated and analyzed for foreign gene in roots
and leaves

13. Pollen tube pathway transformation
technique

14. Transformation in Cotton plant(Gossypium sp.)

15. • Here the time chosen for DNA application was in such way
that the foreign DNA reach the fertilized but undivided egg
so that DNA can interact with genome of single-celled
zygote and produce non-chimeric transformed seed.
✓ APPLICATIONS:-
• Luo and Wu(1989)introduced gene for neomycin
phosphotransferase into rice.
• Zhou et al.(1988) obtained transformed seeds and plants of
rice and cotton.
✓ ADVANTAGES:-
• Simple.
• Efficient and less time consuming.
• Produce non-chimeric transgenic plant.

16. ✓ DISADVANTAGES:-
• Not demonstrated in many species.
• Frequency of transformation is low.
• Operated only in flowering period at blooming stage.
• If use total DNA to transformation,some unwanted gene DNA
segment would be transformed.