Microinjection

This technique had a century of history. This was first described by Dr. Marshall A. Barber, a

professor in bacteriology, in 1902 (Vladimir K, 2002). Microinjection was first applied to isolate

yeast and bacterial cells. In 1985, Lawrence W.A. and Davies D.R. report using microinjection to

regenerate calli from Nicotiana tabacum marked the first microinjection in plant (Lawrence and

Davies, 1985). Microinjection use very fine needles or glass micropipette to inject DNA, always

under the microscope (Ozyigit, I.I, 2020). Protoplasts are main sources in microinjection.

Micromanipulator is required to hold the needle holder. Microneedle is required for penetration

of plant cells and injection. (Thomas C., 2006). Inverted microscope with flat stage is preferred.

Protoplast are immobilized on the agar plate. Fill the microneedle with target DNA. Microneedle

penetrate through the cell membrane and inject DNA into plant cell.

Microinjection is done manually or semi-automatically. Protoplast should be adhered to the

surface such as plastic, glass or agar for immobilization (Gunther and German, 1990), and the

working table is strong enough to minimize vibration, otherwise error occurs. Holding pipette

also used to stabilize protoplast. Micromanipulators attach to microscope (Kunkel T. 2015) and

holds micropipette and move for small distance in three directions, controlled by the user.

microneedles are made from micropipette and puller. Heat the middle of micropipette until

slightly melted, quickly pull capillary apart to form two very fine needles, the tip of needles

can be 10m to 20m, forming microneedle (Tzfira and Hohn, 2013). After that, target

DNA uptake by capillary action into the micropipette (Daniel P., 1982). Microneedle will

penetrate through cell membrane or nuclear membrane. After penetration, injection pressure was

triggered. DNA is injected into cytoplasm or nucleus of plant cells. Sometimes operators will

attach the microneedle with syringe and a pump to facilitate the release of DNA after entering
plant cells. Vacuum is provided to hold the protoplast, fill solution into microneedles if solution

is too viscous, or the microneedle tip is too large to penetrate the plant cell, slight vacuum is used

(Tzfira and Hohn, 2013). Parameters affecting microinjection is injection pressure. If the

pressure is not enough, cell membrane may attach to the microneedles, causing membrane

rapture and apoptosis. Compensation pressure on capillary affect microinjection. If it is too low,

DNA solution will leak out before injection. The injection pressure and compensation should

make a balance to ensure highest transformation efficiency. Moreover, the diameter of

micropipette tip affect microinjection. If the diameter of tip is too large, protoplast mortality rate

is higher, but more DNA can be injected into the cell and vice versa (Joanna H. et al, 2023).

Lastly, cell type, cell size also affects microinjection efficiency.

Microprojectile bombardment (Gene Gun)

Gene gun method was invented by Dr. John C. Sanford, Dr. Edward Wolf and Nelson Allen in

1980s, they used to design a gun like BB gun to shot DNA into cells (Vasil I. K., 2008). Dr.

Sanford described this method as “Biolistic”, which is the fusion of “biology” and “ballistics”. In

1987, Dr. Sanford published a report on Nature, indicate the first stable genetic transformation on

tobacco. Scientist also called this method as “microprojectile bombardment” or “particle

bombardment”. Different plant tissues such as callus, leaves or intact plant cells can be used in

this method. Before applying gene gun method, gene gun, target DNA and microcarrier should

be prepared. Gene gun needs to be sterilized with 70% ethanol and components inside the gun

such as stopping screen, macrocarrier should be autoclaved. Gene gun should be completely dry

before operation. Conclude from Kimberly N.V. et al (N.A.), Kim Sung Yong (2020) and Jose

R.V. (2005), Common microcarrier of DNA is gold particles or tungsten particles. Prepare gold

solution. Add gold particles with isopropanol or ethanol, centrifuge it and remove supernatant.
Add water to resuspend pellet, centrifuge, remove supernatant and repeat 3 times. Resuspend

pellet with 50% glycerol and mix it well. Gold particle solution is prepared. After that, add

plasmid DNA, calcium chloride (CaCl2) and spermidine to gold particle solution, mix

immediately. Add isopropanol to mixture and centrifuge it, leave pellet and resuspend with

isopropanol. Spread mixture to the macrocarrier and ready for bombardment. Target tissue with

the plate is put on plate holder, set parameters such as helium, distance between macro and

microcarrier. DNA coated gold particles are shot to target plant tissue. Place bombarded tissue to

the dark for repairment and transfer to culture medium for cultivation.

Gold and tungsten particles are commonly used in gene gun as they can be ionized to carry

positive charge. DNA carries negative charge because of the phosphate group. An electrostatic

interaction is created between gold and DNA (Godbey W. T., 2022). Spermidine carries positive

charge, facilitate DNA binding to microparticles and CaCl2 can cause DNA precipitation.

Precipitation of DNA can degradation of DNA by intracellular nuclease, so DNA can stay in the

cytoplasm for a longer time to allow DNA uptake by the nucleus (Klein, T. M et al, 1988),

therefore enhance the transformation efficiency. Also, gold and tungsten particles are harmless to

plant cells and these particles can take up DNA easily. DNA coated particles are placed evenly on

the macroprojectile of gene gun. Some gene gun will provide partial vacuum. Gene gun

composes of a gas inlet or gas acceleration tube, connecting to high pressure helium gas. Gas

pressure accumulate on the rupture disc until reaching certain value, the disc breaks and create a

shock wave. (Gozde and Markita, 2018). Macroprojectile is below the rupture disc where DNA

coated particles bind on it. Shock wave generated by helium gas will push the macroprojectile to

the stopping screen. Stopping screen acts as a filter to prevent large size molecules passing

through (large molecule will cause damage to plant tissue) and allow equal distribution of
particles before entering the plant tissue. After that, accelerated particles will propel through the

stopping screen and reach the target plant tissue or cells. High speed particles can penetrate

through the cell wall, cell membrane or nucleus membrane. After entering plant cell, DNA will

dissociate from gold or tungsten particles and integrate with host genome.

Maintaining velocity of microprojectiles is the most important. Type of gas can affect particles’

velocity. Helium gas is commonly used as it creates high compressibility factor and non-toxic to

cells (Marrion M. C. et al, 2005). Compressed gas is another substitute for helium gas. Also,

particle size and density affect velocity. Gold or tungsten particles used in this method ranged

from 1 to 5m, some can be smaller than 1m. Smaller size with higher density like gold

particles have a higher velocity and cause less damage to cells during penetration. Large particles

require stronger gas pressure, cause severe damage to cells or cannot pass through barrier (Zhang

Dong. Wei. et al, 2013). Vacuum can reduce air resistance to microprojectiles and air loss during

pressurization, particles can maintain speed and penetrate deeper into the plant cells (Shashi and

Daniel, 1997). Distance between macroprojectile and stopping screen is another critical

parameter. The distance needs to be set accurately. The speed of microparticles is reduced if the

flight distance is too long, particles do not have enough force to penetrate through the barrier of

target plant tissue, reduce transformation efficiency. Conversely, velocity of particles is too fast

as the distance is too short, the damage of target tissue will be higher (Fatemeh M. et al, 2014).

Lastly, type or size of target plant cells or tissues also affect efficiency of particle bombardment

method.

Nanoparticle-mediated plant transformation

The concept of “nanotechnology” was first introduced by Richard Feynman in 1959, he was

called “the father of nanotechnology”. In 1974, Norio Taniguchi apply and define
nanotechnology as “processing of separation, consolidation and deformation of materials by one

atom or one molecule” (Samer Bayda et al, 2019). After that, nanotechnology has been applied to

various categories. Plant scientists apply nanotechnology to deliver desired gene into plant cells.

Nanoparticles (NPs) are particles in nanoscales with at least one dimension, size ranged from 1

to 200nm, general particle size is 1 to 100nm, magnetic nanoparticles are 200nm in size (Lv Z. Y.

et al, 2020). Before transformation, nanoparticles, desired DNA and target plant tissue (callus,

leaves) should be prepared. DNA can be treated with CaCl2 or spermidine before mixing with

nanoparticles solution. Mix nanoparticles with DNA together by inverting the tube up and down

every 30 minutes, leave it for 2 hours (still searching) depends on experimental condition allow

binding of DNA and nanoparticles. Do not vortex the tube because this will break nanoparticles

and destroy DNA. After that, mix target cells with nanoparticles-DNA mixture and incubate for a

few hours, allow nanoparticles enter the plant cells. Incubate transformed tissue for few days to

allow integration of DNA into genome. Some experiments may require repeat cultivation with

nanoparticles to enhance transformation efficiency.

There are different types of nanoparticles such as carbon nanotubule, DNA nanostructure,

peptide nanoparticles. I will explain more about advantages, disadvantages and applications of

different nanoparticles in discussion part. Nanoparticles are mostly positive charged, called

“cationic nanoparticles”. This allows the formation of NP-DNA complex by electrostatic

interaction between negative charge DNA and positive charge nanoparticles. The cell membrane

is negatively charged as well, nanoparticles can bind to membrane easily by electrostatic

interaction as well, facilitate easier nanoparticles’ uptake (Jin, Sha et al, 2009). Same as gene

gun, DNA are mixed with CaCl2 or spermidine to facilitate DNA precipitation and DNA binding

(Sembada and Lenggoro, 2024). There are two types of DNA binding to nanoparticles. First is
like gene gun, DNA coated to surface of nanoparticles then enter the cell. Second is DNA

packaged into nanoparticles; nanoparticles can act as a protective layer to prevent DNA

degradation by nuclease in the cell. There are different methods of uptake. For plant cell level,

nanoparticles size smaller than 20nm can pass through nanopores on plant cell wall (Yan et al,

2022). The most common nanoparticle uptake is receptor-mediated endocytosis. Some

nanoparticles contain a ligand, which will bind to receptors on the cell membrane, triggering

conformational change of membrane, forming an endosome. Endosome will further fold to

become a vesicle followed by internal transfer (Dario Manzanares et al, 2020). Some

nanoparticles are small enough to pass the membrane through carrier-protein or ion channels. In

plant tissue level, there are different transportation mechanisms of nanoparticles in plant. By

soaking leaves in solution containing nanoparticles or by spraying, nanoparticles can uptake

through stomata. However, the size exclusion limit of stomata is still unknown as it changes all

the time. Waxy cuticle is another path of nanoparticles’ entry. There are two channels on cuticle,

one is hydrophilic channel, allow hydrophilic nanoparticles to diffuse through. Another one is

lipophilic channel, allowing diffusion of lipophilic nanoparticles (Wang Xueran et al, 2023).

Nanoparticles can diffuse through mesophyll cells to enter xylem and phloem and transport to

other plant cells. Uptake of nanoparticles occur in roots as well. Root tissues are soaked in

solution containing nanoparticles or mix nanoparticles with soil. The root surface is negatively

charged, allow binding of positively charged nanoparticles by electrostatic interactions.

Nanoparticles can diffuse into root hair cells and transport through two different pathways. First

is apoplastic pathway, nanoparticles transport through cell wall. Another is symplastic pathway,

nanoparticles transport inside cells and plasmodesmata. Nanoparticles can enter the xylem or

phloem after two pathways and transport to different part of the plant. There are different types
of nanoparticles used as a vector to transport DNA into the plant. Different scientists have

different way to categorize nanoparticles. Cunningham F. J. (2018) categorized nanoparticles into

five classes as shown in Table 1. NPs can be made up of carbon, polymer, metals. Scientists

mainly apply positive charge to surface of NPs, allowing electrostatic interaction between DNA

and nanoparticles.

Table 1: types of nanoparticles and description

Types of Brief description

nanoparticle

Magnetic Metallic nanoparticles are NPs made up of gold, silver, etc. These materials

nanoparticle have good biocompatibility or binding ability with DNA. DNA always

s (MNP) or coated on the metallic NPs due to the electrostatic interactions. Some DNA

metallic will be packed into the NPs (Sharma A. R. et al, 2022). Magnetic

nanoparticle nanoparticles are synthesized by magnetic materials such as iron. MNPs

s can be synthesized in three ways. Physical method is “top down” or

“bottom up” approach. Chemical method includes coprecipitation.

Biological synthesis of MNPs in virus or bacteria. The movement of MNPs

is totally dependent on the application of magnetic field. This property

facilitates the target gene delivery to specific cells by MNPs (Arbab Ali et

al, 2021).

Carbon- Different types of NPs are made up of carbon. Carbon nanotubes are layers

based of graphene sheets, can be divided into single-walled carbon nanotubules

nanoparticle (single graphene sheet, diameter: 1-3 nm) (SWCNT) and muti-walled
s carbon nanotubules (multilayer sheets, outer diameter 2-100nm, inner

diameter 0.4-2nm) (MWCNT) (Mahpara Safdar et al, 2022). They are

modified and adding polyethylene glycol (PEG) to express a positive

charge, DNA can anchor to side wall of NT by electrostatic interactions.

SWNT can be modified by polyethyleneimine (PEI), allowing electrostatic

bonding formation with DNA and protection from nuclease in plant cells

(Yong Yan et al, 2022). Qiaoling Liu et al (2009) show that SWCNT can

penetrate through cell wall and membrane passively due to their small size.

NTs can be over 100nm long, it can carry lots of desired gene into target

cells passively. MWCNTs can penetrate through the nuclear membrane in

the protoplast (Maged F. Serag et al, 2011).

Polymeric Polymeric nanoparticles (PNPs) make up of polymers such as Polyethylene

nanoparticle Glycol, Polyethylene Imine. Cationic ability of polymer allows DNA

s binding through electrostatic interactions. PNPs-DNA is synthesized by

solvent evaporation, nanoprecipitation technique, etc (Raj Rai et al, 2019).

Bio-inspired Calcium phosphate nanoparticles (CaP NPs). This NPs can provide strong

nanoparticle binding force with DNA due to high surface charge. It can penetrate

s through the cell membrane like viral capsids because of its large surface

charge density. Olton Dye et al (2011) reports CaP NPs pass through

membrane passively by clathrin mediated endocytosis. Chitosan

nanoparticles also have the ability of encapsulating DNA inside NPs to

protect against nuclease degradation (Mao Hai Quan et al, 2001). Some

chitosan will conjugate with PEG or PEI to improve encapsulation property

endocytosis (Karayianni M. et al, 2023).

Silicon- This include silica nanospheres, silicon carbide and mesoporous silicon

based nanoparticles (MSNs). Silica has advantages of easy control on size, shape

nanoparticle and high biocompatibility. Most scientist investigate the function of MSNs.

s MSNs are synthesized from tetrathylorthosilicate, with hydrolysis by

ammonia, condensation by ethanol and water hydrolysis. Another method

of synthesis is using surfactant as a template to control the size and shape of

MSNs. Diameter of MSNs is between 2 to 50nm

Chemical method

Polyethylene glycol (PEG) mediated plant genetic transformation

Other than physical methods mentioned above, chemicals can be used to facilitate foreign DNA

entry. Polyethylene glycol is a chemical compound composed of repeating ethylene glycol units,

which is water soluble and commonly used in protoplast fusion. Scientists discovered that

chemical reaction caused by PEG can facilitate DNA entry. PEG aid in the introduction of DNA

into protoplast. Most PEG mediated plant transformation share a similar experimental protocol.

Firstly, protoplasts are isolated and foreign DNA of interest is prepared. Prepare polyethylene

glycol solution with divalent ions such as calcium ions or magnesium ions. Next, protoplast

suspensions and foreign DNA are combined, mix well. Add PEG solution containing divalent

ions slowly or in dropwise into protoplast-DNA mixture. Incubate on ice for around 20-30

minutes, allow DNA entry. Add washing solution to dilute the concentration of PEG in mixture.

Finally, centrifugation is done to collect the protoplasts. Protoplasts are cultured in a specific
medium containing hormones for cell division and differentiation. Transgenic plant is grown.

Some protocols may transfer DNA with selection marker gene into protoplast. Callus tissues

grown from protoplasts will be transferred to selective medium for the selection of successful

transformants.

There are different parameters affecting transformation efficiency. First is PEG concentration and

molecular weight. There are PEG solutions with different molecular weight, such as PEG2000,

PEG4000. The concentration required is strongly related to molecular weight of PEG. If

concentration is too low, membrane permeability is insufficient for foreign DNA to enter the

protoplasts (Lim, F.H. et al, 2021). If the concentration is too high, the protoplast membrane will

be unstable, affecting the regeneration efficiency. Too high concentration of PEG cause over-

precipitation and crystallization of protoplasts, blocking the entry of DNA into protoplasts

(Hayashimoto, Li and Murai, 1990). Also, excess concentration of PEG cause release of DNA

from protoplasts because the membrane permeability is too high. Moreover, the mortality rate of

protoplast is high due to toxicity in high concentration of PEG. The pH is another parameter. pH

has strong relationship with aggregates formation. Maas C., Werr W. et al (1989) examine

mechanisms and parameters affecting PEG mediated transfection of maize protoplast. They

found that high pH (pH 9) cause formation of uneven size and larger aggregates of DNA, this

affects the transformation efficiency as large aggregates cannot enter the protoplasts. They

claimed that pH 6 cause DNA aggregation in fine and homogenous size, enhance transformation

efficiency. Moreover, the incubation time must be carefully calculated. If the incubation time is

too long, DNA will be exposed to nuclease originated from the protoplasts (or the environment),

resulting in DNA degradation. DNA will be released from disintegrated protoplasts, resulting in

low transformation efficiency. If the incubation time is too short, not enough time for clumping
action of protoplasts, cannot trap DNA, resulting in low efficiency as well (Lim F. H. et al, 2021

and Maas C., Werr W. et al, 1989).

Polyethylene glycol can interact with membrane bilipid layer, increasing membrane permeability

by pore formation (Hashizaki K, 2003). However, there are less research on how PEG interacts

with protoplast membrane, further investigation is required. Polyethylene glycol cannot

precipitate DNA itself. Divalent ions are important playmaker in PEG mediated transfection.

Calcium ions or magnesium ions combine with PEG, cause precipitation of DNA. Because DNA

hydration is strongly reduced, water molecules cannot interact with DNA, causing DNA

precipitation. Rapid precipitation allows protection of foreign DNA from nucleolytic activity.

Divalent ions have a positive charge, which can interact with negatively charge DNA by

electrostatic interactions, facilitate better DNA delivery. Calcium ions involved in pore or

channel formation on the protoplast membrane, allow DNA entry (Lim F. H. et al, 2021). Also,

protoplast will clump together in PEG solution with ions, which can trap foreign DNA

precipitate and allow DNA entry. Murakawa T. et al (2008) mentioned that protoplast will be in

“competent state” after adding magnesium ions and PEG. However, adding large volume of

concentrated PEG solution will cause a sudden increase in osmotic potential in the protoplast.

The water potential inside the protoplast will decrease suddenly and lower than the surrounding,

water outside will rush in quickly by osmosis, protoplast will burst and die. So, PEG solution

will be added gradually or in dropwise, followed by continue mixing to allow better control on

osmotic potential of protoplasts and find the optimal concentration of PEG required. Most

experimental protocol will incubate protoplast in ice after adding PEG solution because

protoplasts will clump in low temperature. Using wash buffer to dilute the PEG solution after
transformation allow better recovery and membrane reseal of the protoplast. Some will give a

heat shock to the protoplasts, allow DNA entry.

Biological method

Agrobacterium mediated plant transformation

Agrobacterium tumefaciens and Agrobacterium rhizogenes are gram negative soil pathogenic

bacteria which can transfer part of the Ti or Ri plasmid called T-DNA to plant cells, causing

crown gall disease or hairy root disease. They are the main focus in the earliest plant genetic

transformation research. Since 1890, “crown gall” is used to describe outgrowth in crown roots.

Further research found that there were bacteria causing crown gall formation. Erwin Smith

named it as Bacterium tumefaciens, this name is re-classified as Agrobacterium tumefaciens

(Somssich M., 2019). Agrobacterium is the most well-known genus used as a vector to transfer

foreign genes into plant cells.

Agrobacterium tumefaciens contains a plasmid called Ti-plasmid. Different components in the

Ti-plasmid are important for T-DNA transfer. Ti-plasmid contains virulence gene which aid in T-

DNA transfer and integration, an opine catabolism gene to utilize opine produced by host cells as

the nutrient source. In Agrobacterium mediated transfer, T-DNA will be released from the Ti-

plasmid and transfer to the host cell. Natural T-DNA contains left and right border repeat

sequence, both are 25kb in length which used to define T-DNA region. There is tms gene which

is responsible for auxins overproduction by encoding iaaH and iaaM, enzyme encoding auxins.

The tmr gene responsible for cytokinin overproduction by encoding ipt (isopentenyl transferase,

an important enzyme for cytokinin production). Both auxin and cytokinin promotes cell division

and elongation. The tml gene cause tumor growth. Also, there is an opine gene, encode for
octopine synthase, which cause the host cell to synthesize opine as a nutrient source of

Agrobacterium. Scientist will disarm the T-DNA region by removing tms, tmr, tml and octopine

synthase gene, because scientist aims to transfer foreign genes into plant using Ti-plasmid as

vector, not investigating crown gall disease. Also, scientists can have more space to insert foreign

DNA into T-DNA region.

Agrobacterium rhizogenes is another Agrobacterium species that can transfer part of its DNA to

plant genome. This species causes hairy root disease in plant. It contains a plasmid called Ri-

plasmid. Components in Ri-plasmid is the same as Ti-plasmid. Biggest difference is in T-DNA of

Ri-plasmid. T-DNA in Ri-plasmid contains left and right border sequences, auxin, cytokinin

synthesizing genes, opine synthesizing genes and different Rol genes. rolA can induce bushy

plants and reduced hormonal content in plants such as auxin (aux1, aux2), cytokinin. rolB is

essential for hairy root and meristem formation. Together with rolC, secondary metabolism is

strongly induced in transgenic plant. rolC actively participated in hairy root formation together

with rolA and rolB (Pavlova, O. [Link] al, 2014). rolD is a proline-synthesizing enzyme which

involved in elongation of root (Trovato, M. et al, 2018). Similarly, scientists may disarm the T-

DNA by removing all rol genes, hormone and opine synthesis gene to facilitate easier insertion

of foreign genes. However, if scientists desire to use transgenic plants to produce pharmaceutical

compound, all genes will remain in T-DNA because they are essential for increasing secondary

metabolites production. The mechanism of T-DNA transfer from Agrobacterium rhizogenes is

the same as Agrobacterium tumefaciens.

There are lots of experimental protocols about transferring foreign genes using Agrobacterium.

General steps are concluded. First, explants such as cells, tissues, seeds are prepared.

Agrobacterium used for transformation is isolated. After that, disarm the T-DNA and use the
same restriction enzyme to cut the T-DNA and gene of interest, create complementary sticky

ends. The gene of interest is inserted alongside with selection marker gene which facilitate

selection after transformation. Use DNA ligase to ligate the foreign DNA onto the T-DNA and

transform the genetically engineered Ti-plasmid back to Agrobacterium. Co-cultivate

Agrobacterium with plant explants. Acetosyringone is added in the culture medium to increase

transformation efficiency. Next, remove Agrobacterium suspension and grow explants in

selective media. After forming callus, grow it in appropriate media to induce shoot and root

formation, a transgenic plant is produced.

The mechanism of T-DNA transfer is a research focus of plant scientists. A hormone called

acetosyringone will be released from the wound plant cell. Acetosyringone is a phenolic

compound which induce Agrobacterium infection to the plant (Vasil I. K., 2008). Acetosyringone

will bind to VirA protein expressed on the surface of Agrobacterium. VirA is part of VirA-VirG

complex. There are lots of VirA-VirG complex on the surface of Agrobacterium. VirA is a

phenolic receptor (Bahramnejad B. et al, 2019) and a hybrid histidine kinase (Chen Z. et al,

2022). VirA will be auto-phosphorylated and trigger the phosphorylation of VirG protein in

VirA-VirG complex (Jin S. et al, 1990). VirG protein is a signal receiver. After VirG is

phosphorylated, it will activate transcription of all vir genes in the Ti-plasmid, different virulence

proteins are produced. VirD1, VirD2 will recognize the border sequence of T-DNA and cut the T-

DNA out from Ti-plasmid. VirD1 is a site-specific helicase which unwind the double strand T-

DNA. VirD2 is a site-specific endonuclease which cut the T-DNA strand at left and right border,

T-DNA becomes single stranded. VirD2 also covalently bind to the 5’ end or right border of the

T-DNA, forming immature T-DNA complex. Binding of VirD2 can prevent digestion of T-DNA

by exonuclease in the plant cell. VirD2 also guide the movement of T-DNA from Agrobacterium
to plant cell (Hwang H. H. et al, 2017). The immature T-DNA complex leaves the Agrobacterium

through T4SS (type IV secretion system). T4SS is synthesized by 11 VirB and VirD4 proteins,

act as a channel for T-DNA transfer. At the same time, VirE2 enter to the plant cell through

clathrin mediated endocytosis. After immature T-DNA complex enters the plant cells, it is coated

with VirE2, forming VirD2-TDNA-VirD2 complex or mature T-DNA complex. Mature T-DNA

complex travel to the plant nucleus through endoplasmic reticulum. Tzfira and Citovsky (2006)

claims that the cytoskeleton on the plant cell membrane also helps guiding the mature T-DNA

complex to the nucleus. After that, VirE2 will be recognized by VirE2 interacting protein 1

(VIP1). Hwang H. H. et al (2017) proposed that VIP1 is an adaptor between VirE2 and nuclear

import machinery of plant cells. MPK3-mediated phosphorylation of VIP1 is activated, causing

trafficking of mature T-DNA complex into the nucleus through nuclear pore complex. Other than

binding on VirE2, there are endogenous VIP1 in plant cells. VirE2-VIP1 can prevent activation

of plant defense gene by inhibiting endogenous VIP1. VIP1 bind on VirE2 and VIP1 associates

mature T-DNA complex to mononucleosome, a chromatin in plant nucleus (Amilia Pratiwi, R.

and Imam Surya, M., 2020) forming Nucleosome-VIP1-VirE2-TDNA complex. VIP1 is

recognized by VirF, an ubiquitin complex, causing degradation of VIP1 and VirE2 protein. VIP1

binding F-box (VBF) also recognize VIP1, uncoat the T-DNA strand by destabilizing VirE2.

Zaltsman, A. et al (2010) proposed that SCF-E3 complex (SKP1-culin-F-box-E3 ubiquitin ligase

complex) is involved in uncoating the T-DNA and degradation of VirE2 and VIP1. Uncoated T-

DNA then integrate into plant genome. The mechanism of integration does not have an absolute

conclusion. Generally, due to mechanical stress or Agrobacterium infection, plant DNA breaks.

T-DNA will be replicated to become double strand. T-DNA will ligate to the DNA breaks by non-
homologous end joining (NHEJ). NHEJ is an error prone process and T-DNA integration is

random as well which needs further research.

Other than Ti-plasmid in Agrobacterium as a vector to transfer gene of interest into plant cells,

scientists invented the binary vector system. Concluded from Lee, L. Y. & Gelvin, S. B. (2008),

T-DNA and virulence genes are in two separate plasmid vectors. One is helper plasmid, which

contains virulence genes needed for T-DNA transfer, which is introduced into Agrobacterium

first. Another plasmid is called binary vector. Binary vector has the following properties, 1) an

origin of replication which can replicate in both E. coli and Agrobacterium. 2) an antibiotic-

resistance gene which allows the selection of Agrobacterium containing the binary plasmid.

Agrobacterium with binary plasmid will be used in transformation. 3) T-DNA region contains

left and right border sequences which allow recognition by VirD1 and VirD2 proteins, gene of

interest and selection marker gene is inserted between border sequence. The binary vector will be

introduced into Agrobacterium containing the helper plasmid by electroporation or heat shock.

After selection of Agrobacterium with the presence of binary vector, co-cultivate Agrobacterium

with plant cells allow infections, virulence genes in helper plasmid will be activated and transfer

T-DNA region from binary vector to plant cells and integration into plant genome.

There are different methods of Agrobacterium mediated transformation. Other than co-

cultivation of Agrobacterium and plant cells, there are agroinfiltration and floral dipping.

Agroinfiltration is mainly applied on transient transformation in leaves rather than plant cells.

Concluded from Chen, Q. et al, (2013), there are two types of agroinfiltration. First is syringe

infiltration. Agrobacterium carrying gene of interest is mixed with the infiltration buffer and

loaded to the syringe. Use a needle to create scratch or a nick on the leave. Infiltrate the

Agrobacterium through the scratch or nick using syringe. The leaf color will change from bright
green to dark green, indicate Agrobacterium suspension has entered the leaf. Vacuum infiltration

is another type. Leaves will soak into Agrobacterium suspension with infiltration media and

place inside a vacuum chamber. After that, apply negative atmospheric pressure to create a

vacuum environment. Air in interstitial spaces of the leave are pulled out; Agrobacterium

suspension will fill in the air space of the leaf.

Floral dipping is another method in Agrobacterium mediated transformation. Floral means “of

flower”. This method aims to transform flowers of a plant using Agrobacterium. Concluded from

Purwantoro, A. et al (2023), Agrobacterium carrying gene of interest is prepared and in

suspension. Dip the flower buds until it’s completely covered by Agrobacterium suspension.

Dipping process varies from 30s to 90s. After that, covered the floral dip to maintain humid.

Grow the plant normally and allow flowering occurs until producing seeds. Normally, flower is

transformed in this method, seeds produced may contain the gene of interest carried by

Agrobacterium. Germinate seeds on culture medium followed by selection of plants using

antibiotics or selective medium. Transgenic plants are produced.