Plant tissue culture

QUICK REVIEW FOR II/II B. PHARMACY STUDENTSUNIT-III: PLANT TISSUE CULTURE
---------------------------------------------------------------------------------------------------------------------
---------------------------------------------------------------------------------------------------------------------
Nandini Mekala_Notes_Pharmacognosy and Phytochemistry-I(BP405T) B.Pharm
II/IISem_Venkateshwara Institute of Pharmaceutical Sciences, Nalgonds, TS. Email:
hechhu.ramana@gmail.com
PLANT TISSUE CULTURE
CONTENTS:
1. Historical Development of Plant Tissue Culture
2. Nutritional requirements, growth and their maintenance
3. Type of cultures
4. Application of plant tissue cultures tissue in pharmacognosy
5. Edible Vaccines
By
MEKALA NANDINI
Under the Guidance of
M.BHASKAR, M.Pharm. (PhD). & H. RAMANA, PhD.
VENKATESHWARA INSTITUTE OF PHARMACEUTICAL SCIENCES,
NALGONDA, TS.

---------------------------------------------------------------------------------------------------------------------
---------------------------------------------------------------------------------------------------------------------
PLANT TISSUE CULTURE
Introduction:
Plant tissue culture also known as micropropagation mainly used for the plant
propagation. Tissue culture involves culture and maintenance of plant cells invitro in sterile
conditions. That is nutritionally and environmentally supportive conditions. Cultures produced
by tissue cultures have same genotype.
1.HISTORICAL DEVELOPMENT OF PLANTTISSUE CULTURES:
The history of plant tissue cultures is started nearly more than 100 years but application
of these techniques to pharmacy takes place mainly in last 25 years. The history of plant tissue
culture started with Haberlandt’s hypothesis of “Totipotency of plant cells”. For this
Haberlandt’s isolate palisade cells from the leaves and place in Knops salts. The cell alive for up
to one month and increased in size but failed to divide. This led to the development of techniques
for cultivation of cells under defined conditions. This made possible by R. J. Gautheret and
PR.White.
White from tomato root tips and Gautheret from cambial tissue cultures successfully
reported cultures. Later white reported continuous cultivation of undifferentiated callus from
hybrid Nicotina. Gautheret examined the influence of inorganic salt mixture, dextrose, cysteine,
and IAA etc.
Ricker and Wu examined hanging drop cultures of separated cells from a callus of hybrid
tobaccos. Maheshwari and Guha in 1964 produce plants from grains of Datura. This led to the
development of pollen cultures or another cultures. These works showed that isolated
microspores of Tobacco produce complete plants.
The discovery of kinetin by Miller enabled the initiation of callus culture of differentiated
tissues. Skoog and Miller proposed the root-shoot regulation of auxin-cytokinin ratio. Murashige
and Skoog were two scientists worked on the tobacco tissue cultures in 1960s and developed
different media to achieve optimal growth. Recently in 2005 rice genome sequenced under
International rice genome sequencing project.
Thus, in a brief period tissue culture technique have made great progress from
demonstrating the totipotency of different plant cells to application both basic and applied
sciences. Tissue culture in pharmacy evolved as an alternative source for production of

---------------------------------------------------------------------------------------------------------------------
---------------------------------------------------------------------------------------------------------------------
secondary metabolites to traditional agricultural and industrial production. Tissue culture now in
pharmacy targeting the quick production secondary metabolites, propagation endangered
medicinal plants, and exploring the biogenetic capabilities of various cells for production of
secondary metabolites economically.
2.NUTRIONAL REQUIREMENT:
As the isolated explants tissue culture unable to receive nutrients and hormones the
explants act as heterotrophic and hence, the explants act as heterotrophic. Therefore, plant tissue
culture requires:
a. Essential nutrients
b. Carbohydrate source
c. Growth hormone [regulators and vitamins]
The rate of morphogenesis in tissue culture is largely governed by composition of
nutrient medium. The nutritional requirements of each species are different for promoting
optimal growth. The nutritional requirements of each species are different for promoting optimal
growth.
According to the need and purpose of culture components and quantities of nutrient
medium varies from species to species. Murashige and Skoog [MS] were two scientists who
worked on tobacco tissue culture and developed different media to achieve optimal growth.
In the last two decades also, different mediums were developed based on MS media
The basic components of any medium are:
1. Inorganic salts
2. Organic/Carbon source
3. Vitamins
4. Phytohormones
5. Organic supplements
6. Trace elements
1. Inorganic salts
Inorganic salts stimulate explants to form callus and also to cultivate callus. Agar is
added to prepare solid medium of inorganic salts. Among the two important Media, MS medium
is widely used and has high concentration of inorganic salts [nitrates, potassium, and ammonia].

---------------------------------------------------------------------------------------------------------------------
---------------------------------------------------------------------------------------------------------------------
The B5 medium was developed by Gamborg has low concentration of inorganic salts when
compared with MS medium.
2. Organic/Carbon source
Carbon source involved in the production of useful metabolites. Generally, sucrose or
glucose at 2-4 percent is suitable carbon sources. But fructose, maltose, and other sugars also
added. The selection of carbon source depends upon individual species and metabolites required.
In industry in the economic point of view use of inexpensive carbon sources such as molasses is
used.
3. Vitamins
Vitamins like myo-inositol, nicotinic acid, pyridoxine, thiamine etc. are used. Among these
Thiamine [B1] is essential for many plants.
4. Phytohormones
Phytohormones are essential for callus cells to induce and promote morphogenesis [mainly cell
lines] and also metabolite production they are:
I.Auxins
At the Concentration [0.1 to 50] mg/lit are used for to cell division cell elongation, formation of
adventitious roots generally 2,4 Dichlorophenoxyacetic acid [2,4-D], Naphthalene Acetic Acid
[NAA], Indole Acetic Acid [IAA], and Indole Butyric Acid [IBA].
II.Cytokinins
At concentration [0.1to 10] mg/lit are used for shoot induction, cell elongation. Generally,
kinetin or Benzyl adenine is used.
III. Gibberellins
Gibberellins are used for plant regeneration, elongation of internodes and induction of
embryogenesis. Ex: Gibberellic Acid
5. Organic supplements
To meet the metabolic/energy requirements and also to stimulate the growth these are necessary.
These supplements include:
I. Nitrogen: In the form of amino acid like glutamine, asparagine, and nucleotide adenine
II. Organic Acid especially TCA cycle acids like Citrate, Malate, Succinate, Fumarate etc.

---------------------------------------------------------------------------------------------------------------------
---------------------------------------------------------------------------------------------------------------------
III. Complex substances like Yeast extract, malt extract, coconut milk, protein hydrolysis etc. are
used.
6. Trace elements
These act as components of proteins, nucleic acids, coenzymes, photosynthesis and
respiration, cofactors, electron transport chain, energy transfer etc. these are classified into three
categories mainly; Macro elements: The elements required in concentration more than 0.5
mol/lit. Ex: N, P, K, Mg, Ca, S, Cl.
Microelements: These elements required in concentration less than 0.5 mol/lit.
Ex: Fe, Mn, B, Cu, Zn, I, Mo, Co.
3.TYPES OF CULTURES:
Introduction:
The culture starts from the explants i.e. piece of plant parts such as leaves, roots or
specific cell types such as pollen or endosperm. Many factors affect the selection, initiation and
efficiency of tissue cultures. Generally younger, rapid growing are preferable. The different
culture types are Callus culture, Suspension culture, Protoplast cultures, Embryo cultures,
Microspore cultures, Anther cultures and Gene transfer cultures.
I. Callus cultures:
Any living part of the plant under appropriate condition acts as Explant. Explants when
cultured under appropriate conditions like medium, Auxins, Cytokinin’s, and other hormones,
vitamin etc. can produce callus. Callus is unorganized growing and dividing mass of cells during
the callus formation during callus formation some degree of differentiation both in morphology
and metabolism. This also led to loss of photosynthesis.
During the long-term, the culture does not require auxins is known as habituation. Callus
culture often performed in the dark as light encourages differentiation. Callus culture extremely
important in plant biotechnology in the development of roots, shoots, and somatic embryos etc.
II. Cell suspension cultures:
Callus cultures are broadly classified into compact or friable. The friable callus is soft
and breaks apart. The friability of callus is improved by sub culturing. When a cell suspension is
large the exhausted and toxic products build up to inhibitory levels known as stationary phase,
If stationary phase is too long, they will die. So, sub culturing is required.

---------------------------------------------------------------------------------------------------------------------
---------------------------------------------------------------------------------------------------------------------
III. Protoplast cultures:
Protoplasts are plant cells with cell wall removed the two general methods for removal of
cell wall are mechanical and enzymatic used are cellulose and peptones enzyme.
Protoplasts are fragile and easily damaged. Therefore, cultured carefully by organogenesis or
somatic embryogenesis whole plant can be regenerated.
IV. Root cultures:
The root tips of either primary or lateral roots can be cultured in-vitro. This is first achievement
of modern tissue culture but not widely used in transformation studies.
V. Shoot tip and meristem cultures:
The shoot tips of auxiliary or adventitious buds cultured in-vitro the meristematic cells
are very useful for clonal propagation. These types of cultures were used in cereal regeneration.
These are less genotype dependent and more efficient.
VI. Embryo cultures /Somatic embryos:
Both immature and mature embryos can be used as explants. Embryo culture is the most popular
method for monocot plant regeneration.
VII. Microspore cultures:
Pollen containing gametophyte is termed as microspore. By using pollen or anthers as an
explants haploid tissue cultures done both on liquid and solid type.
VIII. Gene transfer cultures:
Inter specific crosses are made to ornamental plants to produce specific growth
characteristics, colors and resistance against diseases. Ploidy manipulation can also be done to
overcome breeding problems.
For gene transferring, knowledge of regeneration, gene transfer, and DNA integration are
used. In gene transfer various markers are used for removal and insertion of genes. Gene
transferred plants are also known as genetically modified [GM] plants. In tissue culture various
pre and post breeding problems are carefully monitored.

---------------------------------------------------------------------------------------------------------------------
---------------------------------------------------------------------------------------------------------------------
4.APPLICATIONS OF PLANT TISSUE CULTURES:
1. Micropropagations are very useful to conserve rare or endangered plant species.
Micropropagation also used to conserve and propagate medicinal plants. Large scale growth of
plant cells in liquid culture are acts as a source of secondary metabolites.
2. Propagation of Virus free plants is possible through meristematic tissue cultures. The
herbicides Monuron and Diuron are useful in callusing especially shoot regeneration from leaf
segments of Choleos and petiole of Alfalfa.
3. Cross pollinating of distantly related species and then tissue culturing results into
embryos production. By using rapid growing suspension cultures large volumes of secondary
metabolites are produced with some degree of morphological [or] biochemical differentiation
and slow growth. By tissue cultures fragrances, flavors, natural sweeteners, pharmaceuticals,
antimicrobials are produced. Tissue culture techniques are independent of various environmental
factors like climate, pests, diseases etc.
4. The extracts from tissue cultures of Black carrot shoots contain Anthocyanin which have
antibreast cancer property. Scopolia, Parbiflora, adventitious roots culture produces enzymes
which are useful in the production Hyoscyanine and scopolamine.
5. By using Gamborg-B5 medium from explants Taxus glossa are used in the production of
Baccatin-III and Paclitaxel. From the callus culture of Chonemorpha, grandiflora by using MS
Media and 2,4-D anticancer alkaloid Camptothecin is produced.
- From callus of solanum tuberosum by using MS media glycoalkaloids are produced.
- From the fungus Pencillium citrinum Qunilonine alkaloids are produced in large
quantities by using tissue cultures. In Atropa belladonna the effect of nitrate
concentration [KNO3] on production of alkaloids Hyoscyamus and scopolamine is
observed.
The plant tissue culture of Catharanthus roseous dimeric alkaloid Vincristine is produced.
which is used as anticancer drug. Seed culture is important in propagation of orchids.
Embryo cultures useful in;
1. Prevention of embryo abortion
2. Shortening of breeding cycle
3. Prevention of early ripening

---------------------------------------------------------------------------------------------------------------------
---------------------------------------------------------------------------------------------------------------------
5.EDIBLE VACCINES:
Edible vaccines are nothing but transgenic plant and animal-based production which
contain agents that trigger an animal’s immune response. In simple terms, edible vaccine is plant
or animal made pharmaceuticals. The concept of edible vaccines was developed by Amtzen in
the 1990s.
In initial tradition vaccines and later development of newer, safer and highly effective
vaccines, such as recombinant vaccines, subunit vaccines, peptide vaccines DNA vaccines or
developed but these vaccines have storage and transport problem and also expensive. So, an
alternate cheap, safe and efficient production system for vaccine is required, then the concept of
edible vaccines arises.
Mechanism of vaccines:
Edible vaccines contain DNA fragments from the original pathogen. These fragments
code for a protein that is usually a surface protein of the pathogen, this is responsible for
activation of the body’s immune response.
Advantages:
- These are cheap, these can produce in large quantity.
- These can be ingested by eating plant / part of plant, so purification is not required.
- Extensive storage facilities like deep freezer cold storage are not required.
- Edible vaccines can be produced locally, so need of transportation and distribution can
be eliminated.
- Edible vaccines trigger immunity at mucosal surface, which is first line of defense.
Disadvantages of edible vaccines:
- Surviving ability of antigen to hostile, acidic conditions of stomach in questionable.
- When person came in contact with actual Pathogens body response to it is questionable.
- Dosing of vaccine cannot be controlled.
- There is no separate dosing is available for children and adults.
- The over dosing may responsible for actual diseases.
- Glycosylation pattern in plants and animals are different which may affect the
functionality of the vaccine.
- People may develop allergy to fruit or vegetable containing vaccines.

---------------------------------------------------------------------------------------------------------------------
---------------------------------------------------------------------------------------------------------------------
Examples:
-Transgenic potatoes for Diarrhoea:
- B-subunit of E. coli causes Diarrhoea for this edible vaccine is prepared. These potatoes
should be consumed raw, because the B-subunit is a thermolabile.
- Transgenic tomatoes against Diarrohoea;
For Norwalk Virus, which causes Diarrohea, transgenic tomatoes are developed.
Conclusion:
The first trial on humans with heat liable B-toxin from E. coli is a mile stone in edible
vaccine.
This creates a way to in expensive edible vaccines availability in developing countries
without any refrigerated conditions.
------------------------------------BEST OF LUCK---------------------------------

Plant tissue culture

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Plant tissue culture

Similar to Plant tissue culture (20)

Recently uploaded

Recently uploaded (20)

Plant tissue culture