Plant transformation methods:

I. Indirect Methods
Agrobacterium-mediated (Biological) (Stable)
Viral Transduction (Biological) (Transient)
Inplanta transformation (Stable)

II. Direct method (Stable)

Physical Methods
Chemical Methods
Chloroplast transformation

Transformation methods: 1. Stable transformation 2. Transient expression

Stable transformation-Foreign DNA is integrated with host genome and it is inherited.
Different methods were used to transfer the genes to nuclear/chloroplast genome
Direct gene transfer methods -Chemical methods (PEG mediated) Physical Method-Particle
bombardment, Electroporation, microinjection
Indirect method Biological method (Agrobacterium mediated transformation). The
Agrobacterium system was historically the first successful plant transformation system, marking
the breakthrough in plant Genetic engineering in 1983. The Agrobacterium is naturally occurring
gram negative soil bacterium with two common species A Tumifacience and A rhizogenes. These
are known as natural genetic engineers for their ability to transform plants. A tumifacience induces
tumers called crown galls, whereas A rhizogenes causes hairy root diseases. Large plasmids in these
bacteria are called tumer inducing (Ti plasmid) and root inducing (Ri plasmid) plasmid
respectively. The Ti plasmid has two major segments of interest in transformation that is T DNA
(Transfer DNA) and virulence region. The T DNA region of the Ti plasmid bordered by Left and
Right border (LB & RB) is transferred to plant cell and incorporated into nuclear genome of cells.
The transfer of T DNA is mediated by genes in the region of Ti plasmid called vir genes (virulence
genes). These genes are not transformed to plant genome but necessary for transferring T-DNA
region. Modified Ti plasmid are constructed that lack undesirable Ti genes but contain a foreign
gene (resistant to a disease etc) and a closely linked selectable marker gene (Eg:- for Kanamycine
resistance). Any gene placed within the T DNA region (between left and right border) of plasmid
is transferred to the plant genome. The T DNA is generally integrated in low copy number per cell.
Transfer of gene through wounded plant organs A. tumifacience has limited range of host. It can
infect about 60% gymnosperms and Angiosperm. Hence Agrobacterium mediated transformation
is the method of choice in dicotyledonous plant species, where plant regeneration system are well
established, However, Monocotyledons could not be successfully utilized for Agrobacterium
mediated gene transfer.

Advantages
• It is a natural means of gene transfer; Agrobacterium is capable of infecting plant cells, tissue
and organs; Agrobacterium is capable of transfer of large fragments of DNA very efficiently;
Integration of T DNA is a relatively precise process; The stability of gene transferred is excellent.
Gene copy number is low.
Limitations
• Host specificity – Infect only dicot plants; Somaclonal variation during regeneration of plants;
Slow regeneration; Inability to transfer multiple genes
Steps involved in agrobacterium mediated gene transfer: Identify the suitable explants. CO-
cultivate with Agrobacterium carrying gene of interest. Kill the Agrobacterium
17
(Carbencillin/Cefotaxamine ). Select for transformed cell (Kanamycine). Regenerate whole
plant

Inplanta transformation: It is method of transformation without tissue culture. In this method

Arabidopsis plants at the early stages of flowering were uprooted and placed en masse into a bell jar in
a solution ofAgrobacterium. A vacuum was applied and then released, causing air trapped within the
plant to bubble off and be replaced with the Agrobacterium solution. Plants were transplanted back to
soil, grown to seed, and in the next generation stably transformed lines could be selected using the
antibiotic or herbicide appropriate for the selectable marker gene. Transformation rates often exceeded
1% of the seeds tested. Variations of this extremely simple methods have been widely adopted by

18
Arabidopsis researchers and extended to other crops. Tissue culture and plant regeneration are no longer
necessary and the associated high rates of mutation are avoided.
Transient transformation: In this method of transformation foreign DNA is not inherited. It
is a rapid method and avoids horizontal gene transfer. Two approaches

1. Virus based vectors- Virus cause systemic infection. Tobacco Mosaic Virus (TMV) based
vectors are most commonly used. Other viral vectors are cowpea mosaic virus (CMV) Alfalfa
Mosaic Virus (AIMV) and Potato Virus X (PVX). Antigenic peptide is fused with the viral
capsid protein. Advantage of this method is high level accumulation of capsid protein. Epitope
displayed on surface, self-assembly in to particle and easy to purify. Advantages of virus
mediated transformation is High efficiency of gene transfer. Infection is systemic (Spread to
entire plant). High gene copy number. Vectors used are Non integrative (Gene of interest is
not integrated with host genome)
Virus mediated transformation for vaccine could be done in two methods

1. Expression of full-length recombinant protein. TMV and PVX are commonly used for
this purpose
2. Presentation of foreign epitope on viral particles (VLP). For this method commonly
used viruses are CPMV and AlMV. Here candidate vaccine protein is produced as
part of viral capsid protein. Purified Virus Like Particle (VLP) is used as vaccine

Ideal properties of virus-based vectors for transient expression: It should have broad host range.
Option to carry additional genetic information. Vector should be infectious after genetic
manipulation.
Calimovirus group – CaMV: It is a double stranded DNA Virus. Nacked DNA is infectious.
High copy number 106 virion/cell. Spread entire plant in 3-4 weeks. Limited manipulation i.e.
upto 500 bp DNA can be inserted to vector. It infects only crucifers. Common restriction sites
present on virus genome is disadvantageous.
Gemini virus - Maize streak virus is an example in this group. It infects many agricultural
crops. It is a single strand DNA virus. Coat protein gene can be replaced with foreign gene.
It is transmitted by insects.
Tobomovirus- Tobacco Mosaic Virus (TMV) It is an RNA virus. It multiplies in cytoplasm.
It is most commonly used virus for transient transformation.

2. Transient transformation by Agroiunfiltration/Vaccum infiltration: Whole plant/part

of the plant is infiltrated with agrobacterium containing vector with gene of interest. Every
cell is infected with agrobacterium and it delivers the gene. Gene enters the nucleus and may
or may not integrated to genome. This method can be used to test the efficacy of the protein
before going for stable transformation. Newcastle Disease Virus protein (HN Glycoprotein)
was expressed by agroinfiltration in Nicotiana benthamiana.
Parameter Stable transformation Transient transformation
Expression Whole plant Local or systemic
Time required Long (Months) Short (Days)
Horizontal gene transfer Yes No
Expression level Low High
Method Transformation Virus or Agro infection
19
New transformation method (Magnifection): It is combination of agroinfiltration and Virus
infection (Transduction). Viral vector harboring gene of interest is placed between right and
left boarders of agrobacterium vector. In this method initial delivery of gene into cell is by
agrobacterium and further spread of infection into whole plant is by viral vector. This method
reduces the production cost and time. Various viral vectors have been developed for small- or
medium-scale protein production. For example, highly efficient, bean yellow dwarf virus
(BeYDV) based single-vector DNA replicon system, which incorporated multiple DNA
replicon cassettes was developed. This vector was able to produce 0.5 mg of antibody per gram
leaf (fresh weight) in tobacco leaves within four days following infiltration.

Vector for magnifection: npt II - encoding nptII gene for kanamycin resistance; LIR - long intergenic
region of BeYDV genome; TEV 5′ - CaMV 35S promoter with tobacco etch virus VSP3′ - soybean
vspB gene 3’ element; SIR (yellow oval) – short intergenic region of BeYDV genome; C1/C2- BeYDV
ORFs C1 and C2, encoding Rep and RepA; RB and LB Right and left border of agro vector

Magnifection Advantages: Very fast production and R&D process - take less than 10 days of
time. Very high expression levels - 5 g of recombinant protein per kilogram of fresh leaf
biomass (up to 80% of the total soluble protein). Inexpensive process – Leaf material and
Bacteria; High protein yield; Low cost of upstream and downstream processing.

20
General scheme for recombinant protein production in plants using magnifection.

Depending on the protein of interest, magnifection can produce up to 0.5–5 g recombinant

protein per kg of leaf biomass. With a green biomass yield of 100–300 ton/hectare per year,
the yield of recombinant protein is expected to be 50–600 kg/hectare/year. The expression time
is 6–10 days, making magnifection especially attractive for those applications where rapid
industrial manufacturing is required. Components (a), (e) and (f) are standard existing
industrial processes. Components (b), (c) and (d) are new elements, because large-scale
infiltration (b) requires special equipment and the use of bacteria in steps (b), (c) and (d)
requires biological containment to prevent the release of genetically engineered bacteria into
the open environment.

Yields of protein obtained from Magnifection: The following proteins were produced using
magnifection 1.2 g/kg for somatotropin, 5.1 g/kg for interferon alfalfa, 5.4 g/kg of several
bacterial antigens, 0.4–1.5 g/kg for various single chain antibodies.

Mapp Biopharmaceutical Inc., using similar methodology, transiently expressed the humanized
antibodies, MB-003 and ZMab, in tobacco leaves. MB-003 and ZMab were later combined and
designated as ZMapp. The use of these antibodies as a pharmaceutical drug was able to cure
100% of Ebola infected rhesus macaques primates. Agrobacterium infiltration of leaves for
transient expression is very lab intensive and is the primary reason that sufficient supplies of
the Ebola vaccine are not available. In September 2014, the U.S Department of Health and
Human Service (HHS) provided a grant of $42.2 million to Mapp Biopharmaceutical Inc. for
developing a method for large-scale production of ZMapp. With further optimization of the
transient expression system, large-scale production in a short time period (one week) may
become feasible.

Particle bombardment: The technique involves coating 1μm diameter particles of tungsten or
gold known as microprojectiles with DNA, which are then accelerated to high speed using a pulse
of high-pressure helium into an evacuated chamber containing the target tissues. These DNA coated
particles penetrate through the cell wall releasing DNA from particles which can express transiently
or get integrated into nuclear genome of that cell. With appropriate tissue culture and selection,

21
transgenic plants can be regenerated. Particle bombardment has been used for transformation of
monocotyledonous crop plants such as maize, rice, wheat etc. High copy number is a problem.
High gene copy number suppresses the gene expression.

Chloroplast as bioreactor:

Chloroplasts are the most general organelles in plants cells and eukaryotic algae. Since the
establishment of the chloroplast transformation system in 1990, chloroplasts have been
considered as an ideal organelle to produce recombinant proteins. Chloroplast transformation
is achieved in many species like soybean, carrot, lettuce, rice and oilseeds. Two different
methods have been tested to produce and target heterologous proteins in chloroplast.

1. To stably insert the foreign gene into nuclear chromosomes and then target the
expressed proteins into chloroplast via transit peptide, a chloroplast targeting signal.
2. To directly target and express the genes into the chloroplast genome (Transplastomics).

Biopharmaceuticals produced in chloroplast are insulin, interferons, and somatotropin.

Advantages:
1. High level of protein expression. Up to 40 percent of total soluble protein was achieved.
Insulin accumulated up to 16% of total soluble protein. This is due to high copy number
of the inserted gene. Each cell contains about 100 chloroplast and each chloroplast
contains 100 chloroplast plasmid accounting 10,000 copy of the inserted gene.
2. High protein stability in the chloroplast.
3. Easy manipulation because, chloroplast genome is very small.
4. No problem of gene silencing
5. Site specific integration of gene into chloroplast genome is possible through
homologous recombination
6. No risk of gene transfer to other plants through pollen. Chloroplast is not present in
pollen

Method of transformation to chloroplast: Gene of interest is transformed to chloroplast by

Biolistic/Particle bombardment method. It involves the introduction of plasmids
containing a gene of interest and marker gene into chloroplasts or plastids. The insertion of
foreign genes into plasmid DNA occurs by homologous recombination via the sequences
flanking at the insertion site. First successful chloroplast transformation was performed in
Chlamydomonas reinhardtii by particle bombardment method. Simple operation and high
transformation efficiency make it a favorable way for plastid or chloroplast transformation.
After the first chloroplast transformation in Chlamydomonas reinhardttii, the stable plastid
transformation has also been established in higher plants, Nicotiana tobacum, Arabidopsis,
rape, rice, potato, lettuce, soybean, cotton, carrot and tomato.
Vector for chloroplast transformation. A vector containing a selectable marker gene (aadA)
under the control of plastid expression signals (promoter, 5 ̍ UTR and 3 ̍ UTR,) flanked by
chloroplast sequences. Homologous recombination takes place between the flanking
targeting arms and recipient plastid genome (plastome). Usually promoter of gene coding
for protein of photosystem II (psbA) is used. Selectable marker gene (aadA) gives
resistance to the antibiotic spectinomycin.

22
 Once the gene is transferred continuous selection is necessary to avoid heteroplasmic and
chimeric cells. Heteroplasmic is the condition where within the chloroplast some of the
chloroplast genome contains gene of interest (Recombinant) and some of the plastid
genomes are nonrecombinant. Chimeric is the situation where some of the cells are having
recombinant plasmid in the chloroplast and other cells are nonrecombinant. By repeated
selection one has to obtain cells which are not heteroplasmic and non-chimeric from which
whole plant is regenerated.

Vector for chloroplast transformation: RE- Regulatory sequence; GOI-Gene of Interest; 3’UTR-
3’Untranslated region; Flanking sequence-is part of chloroplast genome sequence.

Schematic representation of Biopharmaceutical production in plant

23