Gene Manipulation in Culture

Tissue Culture:

Tissue culture is the growth of tissues or cells in an artificial medium separate

from the organism. This is typically facilitated via use of a liquid, semi-solid, or
solid growth medium, such as broth or agar. Tissue culture commonly refers to
the culture of animal cells and tissues, with the more specific term plant tissue
culture being used for plants. The term "tissue culture" was coined by American
pathologist Montrose Thomas.

Gene Manipulation Techniques

1. Conventional
(i) Simple Selection
(ii) Crossing
(iii) Inter-species Crossing
(iv) Somaclonal Variation
(v) In-Vitro Mutagenesis
(vi) Ploidy Manipulations
(vii) Protoplast technology

2. Advance
(i) Microbial Vectors
(ii) Microprojectile Bombardment
(iii) Electroporation
(iv) Microinjection
(v) Crispr
 Conventional
(i) Simple Selection:
The easiest method of plant genetic modification used by our nomadic
ancestors and continuing today, is simple selection. That is, a genetically
heterogeneous population of plants is inspected, and “superior” individuals—
plants with the most desired traits, such as improved yield—are selected for
continued propagation. The others are eaten or discarded.

(ii) Crossing:
This technique involves the crossing of 2 different variants of a plant/animal
having superior traits and getting a final variant which may contain the traits
and features of both the parents more pronounced .

(iii) Inter-species Crossing:

Closely related species are crossed and we get a new individual having traits of
both the parents.

Just like the cross between Tiger and lion resulting in Liger.

(iv) Somaclonal Variation:

Genetic variations in plants that have been produced by plant tissue culture
and can be detected as genetic or phenotypic traits.

This is done by 3 main techniques:

(iv.i) Physiological Cause:

Exposure of culture to plant growth regulators and altering Culture conditions.

(iv.ii) Genetic Cause:

Change in chromosome number aneuploidy

– gain or loss of 1 or more chromosomes polyploidy
– gain or loss of an entire genome translocation
– arms of chromosomes switched inversion
– piece of chromosome inverted

Change in chromosome structure

-Deletion
-Inversion
-Duplication
-Translocation
-Genetic Cause

Change in DNA
-Detection of altered fragment size by using Restriction enzyme

Methylation of DNA
-Methylation inactivates transcription process

(iv.iii) Biochemical Cause:

-Lack of photosynthetic ability due to alteration in carbon metabolism

-Biosynthesis of starch via carotenoid pathway
-Nitrogen metabolism alteration
(v) In-Vitro Mutagenesis:
We use mutagens to alter the DNA.

(i) Chemical Mutagens:

- Base Analogs
- Base altering Chemicals
- Agents Altering DNA Structure

(ii) Physical Mutagens:

-Ionizing radiation

(vi) Ploidy Manipulations:

Polyploidy is the state of a cell or organism having more than two paired
(homologous) sets of chromosomes. Most species whose cells have nuclei
(eukaryotes) are diploid, meaning they have two sets of chromosomes—one
set inherited from each parent. However, some organisms are polyploid, and
polyploidy is especially common in plants. In addition, polyploidy occurs in
some tissues of animals that are otherwise diploid, such as human muscle
tissues. This is known as endopolyploidy. Species whose cells do not have
nuclei, that is, prokaryotes, may be polyploid, as seen in the large bacterium
Epulopiscium fishelsoni. Hence ploidy is defined with respect to a cell. Most
eukaryotes have diploid somatic cells, but produce haploid gametes (eggs and
sperm) by meiosis. A monoploid has only one set of chromosomes, and the
term is usually only applied to cells or organisms that are normally diploid.
(vii) Protoplast Technology:
Protoplasts are the cells of which cell walls are removed and cytoplasmic
membrane is the outermost layer in such cells. Protoplast can be obtained by
specific lytic enzymes to remove cell wall or by mechanical ways.

This is usually done through somatic hybridization.

Somatic Hybridization:

Development of hybrid plants through the fusion of somatic protoplasts of two

different plant species/varieties is called somatic hybridization.

This is non-conventional genetic procedure involving fusion between isolated

protoplast under in-vitro condition and subsequent development of their
products to a hybrid plant.

Advance
(i) Microbial Vectors:

Agrobacterium tumefaciens is a naturally occurring soil microbe best known

for causing crown gall disease on susceptible plant species. It is an unusual
pathogen because when it infects a host, it transfers a portion of its own DNA
into the plant cell. The transferred DNA is stably integrated into the plant DNA,
and the plant then reads and expresses the transferred genes as if they were
its own. The transferred genes direct the production of several substances that
mediate the development of a crown gall. Among these substances is one or
more unusual nonprotein amino acids, called opines. Opines are translocated
throughout the plant, so food developed from crown gall-infected plants will
carry these opines. In the early 1980s strains of Agrobacterium were
developed that lacked the disease-causing genes but maintained the ability to
attach to susceptible plant cells and transfer DNA. By substituting the DNA of
interest for the crown gall disease-causing DNA, scientists derived new strains
of Agrobacterium that deliver and stably integrate specific new genetic
material into the cells of target plant species. If the transformed cell then is
regenerated into a whole fertile plant, all cells in the progeny also carry and
may express the inserted genes. Agrobacterium is a naturally occurring genetic
engineering agent and is responsible for the majority of GE plants in
commercial production.

(ii) Microprojectile Bombardment/Gene Gun:

Klein and colleagues (1987) discovered that naked DNA could be delivered to
plant cells by “shooting” them with microscopic pellets to which DNA had been
adhered. This is a crude but effective physical method of DNA delivery,
especially in species such as corn, rice, and other cereal grains, which
Agrobacterium does not naturally transform.

(iii) Electroporation:
In electroporation, plant protoplasts take up macromolecules from their
surrounding fluid, facilitated by an electrical impulse. Supplying known DNA to
the protoplast culture medium and then applying the electrical pulse
temporarily destabilizes the cell membrane, allowing the DNA to enter the cell.
 (iv) Microinjection:
DNA can be injected directly into anchored cells. Some proportion of these
cells will survive and integrate the injected DNA. However, the process is labor
intensive and inefficient compared with other methods.

(v) Crispr:
Bacteria and Archea Prokaryotes have defense mechanisms against viral and
plasmid cellular invaders, just as multicellular organisms. One of these defense
mechanisms is an adaptive immune system found in many bacteria and most
archea called Clustered Regulatory Interspaced Short Palindromic Repeats or
CRISPR, along with the CRISPR-associated Proteins or Cas proteins. By integrating
DNA sequences that are identical to past invaders into their genome, bacteria and
archea generate a cellular memory of past invaders. These acquired sequences
allow the bacteria or archea to recognize viral or plasmid invaders as non-self,
resulting in the degradation of the invading sequence and functioning as an
adaptive immune system for prokaryotes. CRISPR immunity is characterized by
distinct phases. First, during the adaptation phase bacteria or archaea gain a
cellular memory of the invading virus or plasmid. Short sequences of the viral or
plasmid genomes are integrated into the CRISPR locus of the bacterial or archaeal
genome. These CRISPR loci were first identified by scientists working in the
fermentation industry, where prokaryotes are essential to the production of
fermented products. Through comparative genomic analysis of different S.
thermophilus strains (a microbe used in producing yogurt), scientists identified a
highly variable locus in the genome of these bacteria . This highly variable region
had two distinct features: many non-contiguous repeats that are separated by
variable sequences, termed spacers. Upon closer inspection, researchers found
that the spacer sequences matched those found in phage (viruses that infect
bacteria) genomes. Interestingly, when researchers compared phage resistant and
phage sensitive S. thermophilus, the phage resistant bacteria had spacer
sequences that matched regions of that phage’s genome. Thus, spacer content
correlated with phage resistance leading to the model that short regions of the
invader’s genome are integrated into the CRISPR loci as a spacer, separated by
repeat sequences, resulting in a cellular memory of previous infections. After the
acquisition of spacers, RNA, termed the CRISPR RNA (crRNA), is generated from
spacers at the CRISPR locus and loaded onto a Cas protein. crRNA directs the Cas
protein to recognize invading sequences and cleave the incoming phage or
plasmid DNA . Three different types of CRISPR–Cas systems have been identified
in bacteria and archea: Type I, Type II, and Type III. Each system utilizes a different
mechanism to generate crRNA and Cas proteins that catalyze the nucleic acid
cleavage. Here we will focus on the Type II CRISPR system, which has been most
commonly adapted for genome editing due to its simplicity requiring just one Cas
protein, Cas9, and two RNA components. To generate the crRNA, the CRISPR locus
is transcribed, generating a long RNA molecule with sequences homologous to
past invaders. This RNA molecule is termed the pre-crRNA. A second RNA from a
genomic locus upstream of the CRISPR locus is also transcribed. This RNA is called
the trans-activating CRISPR RNA (tracrRNA). The tracrRNA has a region that is
complementary to the repeat region of the CRISPR locus, and binds to the newly
transcribed pre-crRNA creating a double-stranded RNA which gets cleaved by
RNaseIII (an enzyme that recognizes and cuts double-stranded RNA) resulting in a
crRNA:tracrRNA complex containing just one spacer sequence (Fig. 1B). This RNA
complex then associates with a single Cas9 protein, creating an active
ribonucleoprotein (RNP) complex. Once the crRNA:tracrRNA is Cas9 bound, Cas9
is activated and can cleave invading nucleic acid sequences (interference). Cas9 is
termed an RNA-guided endonuclease: it cleaves DNA at sequences that bind to
the crRNA of the Cas9 RNP. Searching the invading DNA for sequences
complementary to the crRNA occurs through Cas9 binding to sequences in the
invading viral or plasmid genome termed Proto-spacer Adjacent Motifs or PAMs.
Different Cas9 proteins from different species of bacteria or archea recognize
different PAM sites. To date, S. pyogenes Cas9 (SpCas9) which recognizes a 50 -
NGG-30 PAM is the most commonly used for genome editing. Two critical
arginine residues in SpCas9, Arg1333 and Arg1335, interact with the guanine
nucleobases of the PAM on the noncomplementary strand. This interaction
between the guanines of the PAM and the arginines in SpCas9, positions the
phosphate of the DNA backbone 5’ to the PAM to interact with a phosphate-lock
loop in Cas9 and facilitate DNA strand unwinding. If the DNA is complementary to
the guide RNA, an RNA:DNA hybrid forms, called an R loop, and cleavage follows.
DNA cleavage results from the action of two different Cas9 nuclease domains: the
HNH domain nicks the DNA strand that is complementary to the crRNA and the
RuvC-like domain nicks the strand that is not complementary to the crRNA. Cas9
cleaves the DNA 3 base pairs upstream of the PAM, resulting in a blunt-end
cleavage of DNA. Cleaving the DNA is deleterious to the invading plasmid or virus,
resulting in degradation and protection against these invaders.