Microinjection is a technique for introducing DNA into plant cells by directly injecting it through the cell membrane using a microscopic needle. The document describes microinjection and its application in crop improvement. It details the microinjection process, including preparing the needle and holding the cell, precisely injecting the DNA, and recovering transgenic plants. Advantages are precise delivery and ability to introduce various DNA structures. Disadvantages include being costly, limited to protoplasts, and slow. As a case study, cotton plants were transformed via microinjecting a modified cry1Ia gene into flower ovaries, resulting in a transgenic cotton line with the full cry1Ia gene integrated into its genome.

1. MICROINJECTION
AND ITS APPLICATION IN CROP IMPROVEMENT
• Submitted to:
Dr. A. A. Sakure
Assistant Professor
Dept. of Agril. Biotechnology
B. A. College of Agriculture,
AAU, Anand
• Submitted by:
Swapnil Baraskar
Reg. no. 2010119009
Dept. of Genetics and Plant
Breeding
B. A. College of Agriculture,
AAU, Anand

2. WHAT IS MICROINJECTION ?
• Microinjection is the conventional method of introducing DNA
by microscopic examination of the targeted specimen, e.g.,
meristematic cells or any defined compartment of a cell.

3. GENERAL INFORMATION:
• A hundred years ago, Dr. Marshall A. Barber proposed a new technique -
the microinjection technique. He developed this method initially to clone bacteria
and to confirm the germ theory of Koch and Pasteur.
• The microinjection technique is a direct physical approach, and therefore host‐range
independent.
• In this method, protoplasts are required. Hence in case of animals, the cell can be
utilized directly but in case of plants, it has to be digested first to remove its cell
wall to get the protoplast.
• Microinjection assembly consists of a low power stereoscopic dissecting microscope,
and two micromanipulators, one for a glass micropipette to hold the host cell and
other for a glass injection needle to introduce the DNA.
• Transgenic plants, however, were only recovered in several studies involving such
species as tobacco (Schnorf et al., 1991), petunia (Griesbach, 1987), rape (Neuhaus
et al., 1987), and barley (Holm et al., 2000), and usually at very low frequency.

4. STEPS INVOLVED IN THE PROCESS:
• The microinjection needle is made by drawing out a heated glass capillary to
a fine point.
• Glass micropipette are prepared to have openings of about 0.3 µM in
diameter and are inserted into plant cell cytoplasm and nuclei with the aid of
a micromanipulators device.
• This method of gene transfer uses a specialized optical microscope setup
called micromanipulator. Using this setup, the needle is set to be inserted
in the host cell.
• In order to microinject protoplasts or other plant cells, the cells need to be
immobilized. It can be achieved by using a holding pipette, by poly-L-lysin
coated cover slips or by embedding the cells in agarose, agar or sodium
alginate.

6. • Cells to be microinjected are placed in a container
• A holding pipette is placed in the field of view of the microscope. The
holding pipette holds a target cell at the tip when gently sucked.
• The tip of the micropipette is injected through the membrane of the cell.
Contents of the needle are delivered into the cytoplasm and the empty
needle is taken out.

7. ADVANTAGES:
• The amount of DNA delivered per cell is not limited by the technique and
can be
optimized. This improves the chance for integrative transformation
• The delivery is precise, again increasing the chance of
integrative transformation.
• The small structures can be injected containing only a few cells and with
high regeneration potential.
• Since it is a direct physical approach, it is host-range independent.
• It allowed the introduction not only of DNA plasmids but also of whole
chromosomes into plant cells.

8. DISADVANTAGES:
• Costly method and requires high skills.
• Method is limited for protoplasts only.
• Injection can cause damage that affects embryonic survival and can result in
quite high mortalities.
• Only one cell is targeted per injection.
• The procedure is very slow and requires an expensive setup of
micromanipulator.

9. Case study…

11. Procedure:
• In the greenhouse, cotton seeds of four Brazilian cultivars were sown in pots (40 cm diameter) filled with
nutrient-rich soil. Each pot contained only two seedlings.
• At full blooming, 50–60 days after emergence (dae), plants were transformed by the microinjection
technique.
• A 10μL drip of DNA (100 ng μL−1) was introduced onto the ovaries of young bolls (24 h after pollination)
using a glass microcapillary injection pipette.
• All putative transformed seeds (T0 generation) were collected and sown in the greenhouse for further
assays.
• Putative Transgenic Lines (PTL) were analyzed by PCR using three primer combinations.
• A fragment of 0.44 kb (1 F/3R) collected from this line was purified, sequenced and analyzed using BLAST
tools.
• The results showed high homology between the sequences of cry1Ia from BRS 293 T0-34 and the Bt cry1Ia
gene deposited in the NCBI gene bank (DQ535488).
• In order to confirm the number of copies of cry1Ia integrated to the genome of the T0-34 line, Southern blot
assays were carried out.
• Only one hybridization signal was seen at∼5.0 kb, indicating that complete gene construction was
successfully introduced.

12. Gene Construct:
• The gene used in the construction (cry1Ia) was previously isolated from the Bacillus thuringiensis
S1451 strain.
• The original sequence of the cry1Ia gene was edited with 40% changes based on the cotton codon
usage, and a linear cassette (∼3 kb) (Fig. 1) was synthesized (promoter+open reading
frame+terminator.

13. • Only in cv. BRS 293 was at least one PTL (T0-34) identified in all three primer combinations,
indicating possible complete integration of cry1Ia based on PCR reactions.

14. Fig. Blast analysis between the sequences of cry1Ia from BRS 293 T0-34 and the Bt cry1Ia gene deposited in the NCBI
gene bank