SCREENING AND SELECTION

OF
HIGH YIELDING CELL LINES

BY INDER MAKHIJA
MCOPS, MANIPAL UNIVERSITY
 Introduction
  
Plant tissue culture is a good source of
production of secondary metabolites, but plant
tissue culture shows variation in production
of metabolites, hence it is necessary to
screen and select the cells, which yield high
production


Cell lines are the cells of a single type that

are capable of indefinite growth in vitro.
 High yield cell lines
Cell lines yielding higher concentration of
secondary metabolites. All the cells are of
single types. They can be sub- cultured
easily


 Screening
 passive technique by which a great number
of cells alone for a certain trait and those
showing desired feature are further
cultivated and screened.


S e le ctio n

S e le ctio n is a n a ctive p ro ce ss, w h ich

d e lib e ra te ly fa vo rs o n ly th e su rviva lo f th e
d e sire d va ria n t w h ile w ild typ e ce lls a re kille d .

It means picking up those cells which are
screened and show desired trait. Cell culture
are heterogeneous in nature, hence it’s
important to selected isolated high yield
cells
 Reasons for screening

and selection
To get cell lines which are having potential to
yield high quality and quantity of secondary
plant metabolites like alkaloids, glycosides,
fats, tannins, polyphenol, pigments etc which are
limited to certain type of cells.
With automatization of cell growth control and
metabolic processes regulation, cost price can
decrease and production increase.
Negative biological influences that affect
secondary metabolites production in the nature
are eliminated (microorganisms and insects)

 Method of screening and
selection of high yielding
cell lines
 Destructive method
As the name indicated the cells have to die
for the screening of secondary metabolites
 Eg. cell squash method, fluorescent antibody
method etc


Non- destructive method 



This method involved do not involving killing

of cells. E.g. visual selection, microscopic
estimation

 Techniques for screening
and selecting high
yielding cell lines
 1. Chemical or analytical techniques
 a . R a d io im m u n o a ssa y ( R IA ) a n d its m o d ifica tio n
 Flu o re sce n t a n tib o d y
 Flo w - cyto m e tric syste m
 Im m u n o sta in in g p ro ce d u re
 

 b . E LIS A
 c . m icro sp e ctro p h o to m e try
 d . H P LC


2. Cell squash method
 3. U.V. spectroscopy and
fluorescence
 4. Cell permeability method or
adsorbent
 filter methods
 5. Visual analysis
 6. Chemical markers
 7. Cell cloning
 8. Auxotrophic mutant
Chemical or analytical


techniques
a . Radio immuno assay ( RIA )
•
Radio immunoassay was developed in 1959 by
Solomon, Benson and Rosalyn Yalow for
estimation of insulin in human serum

•RIA is used for estimation of natural compounds
and various
hormones and proteins present even in low
concentration

•RIA is highly sensitive and specific methods

 Principle
Radioimmunoassay combines the principles of
radioactivity of isotopes and immunological
reactions of antigens and antibody, hence the
name RIA.
The principle of RIA is primarily based on the
competition between the labeled and
unlabelled antigens to bind with antibody to
form antigen antibody complex. (Either labeled
or unlabelled.)
The unlabelled antigen is the substance says
any natural product to be determined e.g.
quinine.
Application of RIA
 Drug substance like digoxin, digitoxin, morphine


 Steroids


 Hormones


 Vitamins


 Nucleic acid


 Structure of proteins and hormone receptor

proteins
 Modification of Radio immuno assay
(RIA)
  
1. Fluorescent antibody method

 

 The test is based on labeling an antibody molecule with a

fluorescent compound e.g. fluorescein isothiocyanate.
 

 F- Labeled antibody + antigen F- labeled

antibody-antigen complex.


 It an be determined under light at 360 nm. And it can also

determine in the visile light spectrum.
 fluorescence assay are used to select ajmalicine and other major
heteroyohimbine alkaloids from the cell culture of catharanthus
roseus.
 
2. Immunoassaying.

 

3. Flow cytometry.

 b . E L IS A ( e n zy m e lin k e d
im m u n o so rb e n t a ssa y )
  
 ELISA is based on the immunochemical principle of antigen-antibody
reaction.
  
 The antibody against the protein to be determined is fixed on an
inert solid such as polystyrene


 The cell containing protein to be estimated is applied on the

antibody coated surface.


 The protein antibody complex is then reacted with a second protein

specific
antibody to which an enzyme is covantelylinked. These
enzymes must be easily assayable and produce preferably colored
product. Peroxidase, amylase, alkaline phosphate are commonly used.


 After washing the unbound antibody linked enzyme, the enzyme

 c . Microspectrophotometry
  

 Hall and yeoman used microspectrophotometry to demonstrate that

cells accumulating anthocynin differ from the remaining cells in
catharanthus roseus with respect to absorbance spectra.



 A microscopic thin beam of light enters the outer segment of the
receptor. Some of light is absorbed by photo pigment and emerges out
and captured by very sensitive detector. And difference between
incident light and captured light is the amount of light absorbed.



 It is techniques of measuring the light absorbed, reflected and
emitted by microscopic specimen at different wavelength.


d . HPLC
  
 HPLC is good tool for detection of secondary
plant metabolites. E.g. Hypericum perforatum
and Rhodiola rosea for their secondary
metabolites.


 Rauwolfia serpentine callus shows two types of

cells. A) Yellow-green and B) blue-white. HPLC
examination shows that yellow-green shows
more reserpine and blue-white shows 3, 4, 5,
trimethoxy benzoic acid.

 2. Cell squash method
 


 In this method portions of cell clone are squashed on

filter paper and then sprayed with stain specific to a
compound in order to visualize the high producing cell
lines
 E.g. tobacco ( nicotine ) yielding cell lines- pressed
and sprayed with dragendorffs reagent to develop color
 
 


 3.U.V. spectroscopy and
fluorescence
 


 Difference in U.V. absorbance spectra by single cells of

Coleus blumei and Anchusa officinalis suspension
 4. Cell permeability method or
adsorbent filter methods.
  
 Adsorbent filters can be used as tool for selection
of high yielding cells from suspension culture.
 By this technique suspension cultures are plated on
a cellophane sheet attached to activated carbon
coated filter paper, which in turn was placed on the
nutrient medium. Compounds released by the cells will
be adsorbed in localized areas of the charcoal coated
filter paper. After removing the cellophane, the
filter paper can be observed under light to asses the
quality of metabolites released from different
population.
 E.g. taxol produced by Taxus brevifolia is
detected by culture filterat
 5. Visual analysis
  
 This technique involves the screening and selection
of high yield cell lines by visual methods.
 e.g. -carotene in Carrot.
 Shikonin in Lithospermum culture.
  

6. Chemical markers
  
 Some chemical compound like p-fluorophenyl
alanine and fluorinated tryptophan have been used
to select cell lines which forms rosmarinic acid and
indole alkaloids respectively.

 7. Cell cloning
  
 Cloning of cell can be used as tool to explore
natural heterogeneity with in cell culture and in the
selection of high yielding cell lines.


 E.g. cell cloning can be used for selection of Dacus

carota (anthocynin), Lithospermum
erythrorhizon (shikonin), Nicotina tabacum
(nicotine).

 8. Auxotrophic mutant
  
 Auxotrophic mutant are nutritional or deficiency
mutants as they cannot grow on minimal medium due
to block in a biosynthetic pathway. Only if the
lack essential biosynthetic intermediate caused
by the block is overcome by supplementing the
medium with suitable compound would auxotrophs
grow on minimal medium

Eg. Nitrate reductase deficient cell lines have

been isolated and cultures cell of N . tabacum ,
D . inoxia etc.
 9. Resistant selection
  
 Cell lines which accumulate certain compound,
can be
 isolated from large cell population by
selecting for
 resistant certain inhibitory substance. The
inhibitor
 should be toxic analog of required cell
metabolites
Type of resistance whose toxicity can beCulture
Plant reversed
system by
corresponding
physical stress natural
 compound
UV light
Water stress
Damascene
Lycopersicon esculentum
Suspension culture
Suspension culture
eg


 6. Methods of
developing new high
yielding cell line

1.Elicitors
Microbes, their extract and certain chemicals are known
for stimulating particular facets of plant secondary
metabolism is called elicitors
Elicitors induced product accumulation is a novel
approach to obtain secondary metabolites in a large
scale
e.g.(1) increased the amount of catharanthin in cells of
catharanthus roseus in response to inorganic and
inorganic salts
2) Addition of solubilized chitin favours the production of
 2 . G e n e tic tra n sfo rm a tio n
• M a jo r ch a lle n g e s in p la n t b io te ch n o lo g y
co n ce rn th e

tra n sfo rm a tio n o f p la n t ce ll & p la n t u sin g

engineered genes to yield novel plant

constitute
•
•

 By using the plasmid of agrobacterium, the

desired

gene is transfer into plant cell lines &
produces
desired & useful secondary metabolites
3. B io tra n sfo rm a tio n
•One of the recent techniques for the commercial
exploitation of secondary metabolites from cell cultures
is the techniques of biotransformation
•
•Production of valuable plant by biotransformation from
inexpensive precursors that can not be transforming
effectively by chemical or microbial methods is an
interesting area for commercial application of plant
tissue culture. This is the process of chemical conversion
of exogenously supplied substrate by living cells into
desired product

•E.g. the transformation of steviol by cells of stevia

rebaudiana into a stevioside, which is 300 times sweeter
than sucrose, which is used as sweeteners in Japan
A d v a n ta g e o f scre e n in g a n d se le ctio n m e th o d

V a ria n t o r m u ta n t ce lllin eshow

s high yield
–
H ig h q u a lity w ith in le ss tim e

Selected cell can be clonally propagated

More economical for plant have long maturity period

which
e.g:-Papaver brateatum, source of thebanine take two or
three season to rich maturity and lemon plant also
takes four to five
year to give mature fruits
 Application
The selection and screening program is useful for
selecting high

yielding strains. E.g. selection for enhanced activity of

the enzyme in

C.Roseus led increased levels of serotonin

 To understand the physiological basis for the

application of selection

procedure for yield improvement, e.g. nicotiana

 R e fe re n ce s

•Veresham ciddi; “medicinal plant biotechnology”; 4th edition,

pg no. - 475-477

•Irfan khan A.; “Role of biotechnology in medicinal & aromatic

plants”; Vol-I; pg. no – 95-110

•Razdan M.K.; “An introduction to plant tissue culture”; 1st edition;

pg no. - 301-307

•U. Satyanarayana, U.Chakrapani; “Biochemistry”; 3rd edition-2006;

books & allied (p) ltd. Pg no-729-730
TH A N K U
Y O