PLANT TISSUE CULTURE CSIR
Summary
TLDRThis educational video delves into plant tissue culture, a technique crucial for preserving India's rich biodiversity. It outlines the process from sterilization to acclimatization, highlighting its significance in mass propagation, producing disease-free plants, and generating high-value compounds. The video underscores the technique's role in conserving rare species and creating improved crops, showcasing its broad applications in agriculture and biotechnology.
Takeaways
- 🌱 India is home to three of the world's 34 biodiversity hotspots, hosting a vast array of endemic species.
- 🔥 Anthropogenic activities and population growth are threatening the survival of many of these species, leading to a high risk of extinction.
- 🌿 Plant tissue culture is a critical tool for conservation, allowing for the preservation of medicinal plants and meeting the increasing demand for them.
- 📚 The concept of totipotency, introduced in 1902, underpins plant tissue culture, suggesting every plant cell can regenerate into a whole plant.
- 🧪 Plant tissue culture involves growing plant cells, tissues, or organs in sterile conditions on a defined nutrient medium.
- 🔬 The process includes steps like preparing instruments and media, sterilization, inoculation, incubation, and acclimatization of plantlets.
- 🧴 Sterilization of culture media and instruments is crucial, typically done using an autoclave at high temperature and pressure.
- 🌱 Explants, which are parts of plants like leaves, buds, or seeds, are collected from healthy plants and undergo surface sterilization before inoculation.
- 🏥 Inoculation is performed under aseptic conditions in a laminar hood, using sterilized tools to transfer explants to the culture medium.
- 🌡️ Post-inoculation, cultures are incubated under optimal conditions of light, temperature, and humidity to promote growth.
- 🌳 Plant tissue culture is vital for mass propagation, production of disease-free planting material, generation of phytochemicals, and conservation of rare species.
Q & A
What is the significance of India's biodiversity in the context of the world's hotspots?
-India is a mega diverse country with thousands of rare endemic plants and animal species. Out of the total 34 hotspots all over the world, three are in India, indicating its significant role in global biodiversity.
How does anthropogenic activity threaten the species in India's biodiversity hotspots?
-Anthropogenic activities and influx of population have led to the verge of extinction for most of the species in India's biodiversity hotspots, including the extinction of many endemic plant species.
What is plant tissue culture and how does it help in conservation efforts?
-Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues, or organs under sterile conditions on a nutrient culture medium. It helps in conservation efforts by preserving precious plants of medicinal use and overcoming the growing demand for such species.
Who conceptualized the concept of totipotency in plant tissue culture?
-The concept of totipotency, which is fundamental to plant tissue culture, was conceptualized by a German botanist in 1902.
What is the process of micropropagation and how is it used in plant tissue culture?
-Micropropagation is a method used in plant tissue culture to produce clones of plants. It involves various steps such as preparation of instruments and nutrient culture medium, sterilization, inoculation, incubation for growth, and acclimatization of plantlets.
What are the necessary instruments and apparatus required for plant tissue culture?
-The necessary instruments and apparatus for plant tissue culture include forceps, scissors, blade holders, disposable but sterile surgical blades, Petri plates, beakers, Erlenmeyer flasks, test tubes, pipettes, and a bottle with a screw cap.
How is the nutrient culture medium prepared for plant tissue culture?
-The nutrient culture medium for plant tissue culture comprises macro elements, micro elements, vitamins, amino acids, and sucrose. The salts are precisely weighed and dissolved in deionized water, and the pH is adjusted before pouring the medium into flasks or test tubes.
What is the role of autoclaving in the preparation of plant tissue culture?
-Autoclaving is used to sterilize equipment and supplies in plant tissue culture by subjecting them to high-pressure saturated steam at 120 degrees Celsius and a pressure of 15 Pascal for 20 minutes.
What are explants and how are they prepared for inoculation in plant tissue culture?
-Explants are parts of a plant such as petals, leaves, buds, ovaries, seeds, and nodal segments used in plant tissue culture. They are collected from disease-free, healthy, and actively growing plants, cleaned, and surface-sterilized before being used for inoculation.
How is the inoculation process carried out in a laminar hood for plant tissue culture?
-Inoculation in plant tissue culture is done under septic conditions within a laminar hood. The hood is equipped with a high-efficiency particulate air filter, and the explants are sterilized before being transferred into the nutrient medium under aseptic conditions.
What is the purpose of acclimatization in plant tissue culture and how is it done?
-Acclimatization is the process of hardening tissue culture-raised plantlets to outdoor conditions. It involves transferring them to greenhouse or outdoor conditions, where they are subjected to various environmental shocks to prepare them for the harsh conditions outside the controlled in vitro environment.
What are the applications of plant tissue culture in agriculture and conservation?
-Plant tissue culture has applications in mass propagation of plants, generation of quality planting material, production of phytochemicals and high-value products, development of improved and tailor-made crops through transgenic techniques, and conservation of endangered, threatened, and rare plant species.
Outlines
🌿 Introduction to Plant Tissue Culture
India's rich biodiversity is under threat due to human activities and population growth, leading to the extinction of many species. To conserve these valuable plants, particularly those with medicinal uses, scientists employ techniques like plant tissue culture. This method involves growing plant cells, tissues, or organs in sterile conditions on a nutrient medium. The process began with the concept of totipotency in 1902, suggesting that every plant cell can regenerate into a whole plant. The steps involved in plant tissue culture include preparing instruments and culture medium, sterilization, inoculation, plant incubation, and acclimatization before transferring to pots. The required apparatus includes forceps, scissors, Petri plates, beakers, flasks, test tubes, pipettes, and a bottle with a screw cap. All instruments must be cleaned and sterilized before use.
🔬 Sterilization and Media Preparation
Sterilization of glass and plastic layers is crucial for plant tissue culture. This involves autoclaving used cultures and media for an hour, followed by soaking in chromic acid for 16 hours. The items are then rinsed, cleaned with detergent, and washed again before drying in an oven at 60-80°C. The preparation of the culture medium includes a precise mix of macro and micro elements, vitamins, amino acids, and sucrose. The pH is adjusted for optimal nutrient uptake by plants, typically between 5.6 and 5.8. The medium is then poured into Erlenmeyer flasks or test tubes, plugged with cotton, and sterilized in an autoclave at 120°C and 15 Pascal for 20 minutes. This process ensures the sterility of the instruments and the medium without degrading its components.
🌱 Explant Preparation and Inoculation
Explants such as petals, leaves, buds, ovaries, seeds, and nodal segments can be used for plant tissue culture. These are collected from healthy, disease-free plants with high cell activity. After collection, explants are cleaned in water to prevent contamination and browning. They are then surface sterilized in a laboratory using a mild detergent and a fungicide or antibiotic solution. Explants are rinsed with sterile water and transferred to an inoculation chamber. The inoculation process involves transferring the explants into the media under sterile conditions. The chamber is prepared with a laminar hood, which is sterilized with UV light before use. Explants are treated with ethanol and mercury chloride for sterilization, then washed and placed in the culture medium. The inoculated cultures are labeled and incubated under optimal conditions for growth.
🌱 Acclimatization and Applications of Plant Tissue Culture
After incubation, cultures with contaminants are removed, and the remaining are allowed to grow further. Once shoots reach a certain height, they are rooted using auxins in the culture medium. Rooted plantlets are then hardened for acclimatization to outdoor conditions. They are transferred to greenhouses or outdoor environments, where they are subjected to various environmental shocks to adapt from in vitro conditions. After rooting and growth, plantlets are moved to potting mixtures and eventually to outdoor conditions. Plant tissue culture is significant for mass propagation of disease-free plants, generation of quality planting material, production of phytochemicals, and conservation of endangered species. It also aids in the production of improved and tailor-made crops through transgenic methods.
🌱 Conclusion on Plant Tissue Culture
The final paragraph emphasizes the importance of plant tissue culture in various applications, including mass propagation, generation of quality planting material, production of high-value products like pharmaceuticals, cosmetics, and food additives, and conservation of rare and endangered plant species. It also mentions the use of transgenic methods to produce improved and tailor-made crops, such as BT cotton.
Mindmap
Keywords
💡Mega diverse
💡Endemic species
💡Extinction
💡Conservation scientists
💡Plant tissue culture
💡Micropropagation
💡Sterile conditions
💡Explants
💡Acclimatization
💡Endangered species
💡Transgenic crops
Highlights
India's biodiversity is under threat due to anthropogenic activities and population influx.
Three of the world's 34 biodiversity hotspots are in India.
Conservation efforts are crucial for preserving India's endemic plants and animals.
Plant tissue culture is a key technique for preserving medicinal plants and meeting demand.
The concept of totipotency, where every plant cell can regenerate a whole plant, was introduced in 1902.
Plant tissue culture involves growing cells, tissues, or organs in sterile conditions on a nutrient medium.
Micropropagation is a method of cloning plants using plant tissue culture.
The process includes preparation of instruments, nutrient medium, sterilization, and plant incubation.
Instruments for plant tissue culture include forceps, scissors, blades, and various containers.
Sterilization of culture vessels is a critical step to prevent contamination.
Media for plant tissue culture must contain precise amounts of macro and micro elements, vitamins, and sucrose.
Autoclaving is used to sterilize the media and instruments at high temperature and pressure.
Explants from healthy plants are prepared and sterilized before inoculation.
Inoculation is done under sterile conditions in a laminar hood to prevent microbial contamination.
Plantlets are incubated under controlled light, temperature, and humidity conditions.
Acclimatization of plantlets involves gradual exposure to greenhouse or outdoor conditions.
Plant tissue culture has applications in mass propagation, production of high-value compounds, and conservation of rare species.
Transcripts
India is a mega diverse country with
thousands of rare endemic plants and
animal species out of the total 34
hotspots all over the world three are in
India but because of the anthropogenic
activities and influx of population most
of the species are on the verge of
extinction
most of the endemic plant species have
been extinct so far conservation
scientists using various techniques that
help us to preserve these precious
plants of medicinal use and to overcome
the growing demand one of the techniques
is plant tissue culture
today I shall be talking about plant
tissue culture and its applications in
improving crops of importance
the field began way back in 1902 when a
German botanist conceptualized on the
concept of totipotency this concept is
based on the fact that each and every
cell of the plant is capable of giving
rise to an another individual plant and
that plant is as similar or is true as
true as any plant you find in the field
plant tissue culture is a collection of
techniques used to maintain or grow
plant cells tissues or organs under
sterile conditions on a nutrient culture
medium of known composition plant tissue
culture is widely used to produce clones
or plant in a method known as micro
propagation the various steps of plant
tissue culture are preparation of
instruments and nutrient culture medium
sterilization of culture medium
preparation of explant inoculation of X
plant incubation for growth
acclimatization of plantlets and
transfer to pots
preparation of instruments apparatus
required for plant tissue culture are
forceps scissors blade holders
disposable but sterile surgical blades
or sharp scalpel Petri plates with two
filter papers a 500 milliliter beaker 3
250 milliliter Erlenmeyer flasks a 50
milliliter test tube a box containing
disposable pipette tips pipettes and a
thousand milliliter bottle with screw
cap first of all it is important to take
all the required apparatus in a
spotlessly clean condition these items
are then wrapped in newspaper or brown
paper with a knot at the side that has
to be held by hand these are then kept
ready for autoclaving before performing
plant tissue culture experiment it is
required to have clean and sterile
culture vessels before using any
glassware or plastic fares these are
first washed in a four-step process
firstly all use glass and plastic layers
with used cultures and media are
autoclaved for about 1 hour then the
glass and plastic layers are emerged in
a tub full of chromic acid for 16 hours
after this the glass and plastic where's
our first rinsed with water to remove
the chromic acid and then immersed in a
tub full of detergent water and cleaned
using hard nylon brushes
the glass and plastic waves are then
emerged in another tough full of clean
water and then the detergent is rinsed
off finally the glass and plastic layers
are washed under running tap water kept
in clean trays and put in an oven
maintained at 60 to 80 degree Celsius
for drying the dried glass veils are now
ready for use
the next important process is
preparation of a media which is to be
used in the process of micro propagation
a medium for plant tissue culture will
comprise of a group of macro elements a
group of micro elements vitamins amino
acids and most importantly sucrose which
serves as a source of carbon and which
is the carbohydrate for the plant the
amounts of salts to be taken have to be
precisely weighed as per the reported
formulations and these have to be
dissolved in deionized water care has to
be taken to avoid precipitation of salts
as it prevents the availability of salts
to the plants as salts with higher
solubility our first dissolved
adjustment of the pH of the medium
generally plants can take up nutrients
at a pH ranging from five point six to
five point eight after the medium
attains the desired pH it is ready to be
poured into 250 milliliter Erlenmeyer
flasks or into 50 milliliter test tubes
as one hundred milliliter or 20
milliliter alec Watts semi-solid medium
has to be prepared the method followed
is to weigh 0.75 to 0.85 Erica
this amount has to be optimized to
provide optimum moisture to the cultures
pouring of medium 100 milliliter medium
is carefully poured into each 250
milliliter Erlenmeyer flasks and plucked
using cotton plugs made of non absorbent
cotton wool covered with muslin cloth
and tied at the top allowing easy
holding of the plug while inoculation
sterilization of culture medium is
autoclaving an autoclave is a device
used to sterilize equipment and supplies
by subjecting them to high pressure
saturated steam the media prepared and
the different instruments made ready for
use are autoclaved for 20 minutes at
high temperature that is 120 degrees
Celsius and pressure at 15 Pascal
these parameters are just right to
sterilize the instruments and the media
without degrading its composites such as
sucrose etcetera preparation of explants
different kinds of ex plants such as
petals leaves buds ovaries seeds and
Thurs and nodal segments can be used for
plant tissue culture because each and
every cell of the plant is capable of
giving rise to a new individual however
care must be taken to collect these ex
plants from disease free healthy and
actively growing plants preferably
having some meristematic areas of high
cell activity immediately after
collection the ex plants are placed in
clean water to avoid the entrance of air
bubbles microbes and contaminants from
the cut or exposed parts and to avoid
browning due to phenolic oxidation these
ex plants are then brought into a
laboratory for surface sterilization for
this the ex plants are first cut into
smaller size using a sketchier or a pair
of scissors and then place in a petri
plate containing clean water
the surface of the explants is then
brushed clean with a mild detergent such
as tween 20 as a wetting agent with a
sable hairbrush after cleaning the
surface the ex plants are picked up and
dropped into a glass or plastic vessel
containing a mild solution of a
fungicide or an antibiotic the ex plants
are then swirled for a few minutes and
rinsed several times with clean and
sterile deionized water finally the ex
plants are immersed in sterile deionized
water and taken into inoculation chamber
now rx plant is ready for next process
of inoculation prior to entering the
inoculation chamber through the double
door it is important to wear a clean
cotton lab coats and take an Ayrshire
inoculation means transferring plant
specimen into the media under a septic
conditions
preparation of laminar hoods the laminar
hood is a special equipment for
inoculation shut the shutter of the
laminar hood
switch on the UV lights for about 30
minutes while the UV light can kill all
the microbes it is highly dangerous for
human eyes and skin
therefore the shutter has a black sheet
and is not opened until the UV light is
switched off after 30 minutes switching
off of the UV light switch on the
chamber lights open the shutter and then
switch on the air flow prior to
operating the laminar hood it is
essential to wear a clean and sterile
face mask and a cap
the panel of the lamina that faces us
has a high efficiency particulate air
filter the ex plants are washed with 70%
ethanol for a few seconds and then
treated with a mild mercury chloride
solution for a few minutes
a general thumb rule is to use a mild
solution for a longer duration rather
than a strong solution for a shorter
duration after the sterilizing agent
treatment
the solution is decanted into the waste
beaker and the ex plants washed several
times with sterile deionized water to
remove all traces of mercury chloride
all the apparatus are dipped in the 70%
ethanol flamed using the lighted spirit
lamp and allowed to cool
now after cooling the forceps the ex
plants are carefully picked and placed
in the petri plate with filter paper so
that all the excess water content will
be absorbed
surface of the explants which were
exposed to mercury chloride are removed
with the help of surgical blade
each nodal segment is carefully inserted
into the medium contained in the test
tube and care is taken to avoid the ex
plants from touching the rim of the
flask which is again flamed and after
that cap is put back at the mouth to
seal it tightly
finally name of the plant medium and
date of inoculation is labeled onto the
surface of the test tube the cultures
are now ready to be incubated in the
culture lab at optimum conditions of 16
hours light alternating with a tars
darkness 25 to 27 degree Celsius
temperature 40% relative humidity and it
is monitored timely after a few days
which may range from 5 to 15 days all
cultures with either bacterial or fungal
contaminants are removed and the
corresponding cultures are allowed to
grow further
after a period of time one ends up with
a large number of shoots in a single
flask
once the chutes have grown to a certain
appreciable height of about three to
four centimeters they are rooted
generally auxins are used in the culture
medium to induce routing in each of
these shoes once the chutes are routed
these have to be hardened for
acclimatization in the open
now the tissue culture raised plantlets
TCPS are transferred to greenhouse or
outdoor conditions and they are
subjected to different types of shocks
like temperature humidity nutrition
carbon dioxide and airflow shock
the greenhouse and fields have
substantially lower relative humidity
higher light intensity and septic
environment and are therefore stressful
to the TCPS because they have been drawn
out of their comfort zone of in vitro
conditions after rooting and growth of
plant leads up to three to four inches
shift these two other potting mixture
containing garden soil sand and will
decomposed farmyard manure in ratio of 1
is to 1 is to 1
other medium like soil right vermiculite
perlite and coco peat can also be used
for preparation of potting mixture after
45 to 60 days these acclimatized
plantlets can be shifted to the outdoor
conditions these plants are now ready
for the harsh conditions effectively
with very low mortality
importance of plant tissue culture the
process of propagation facilitates the
production of a large number of disease
free quality plantlets independent of
seasons in fact plant tissue culture has
several applications extending from mass
propagation of plants by
micropropagation
or in vitro culture generation of
quality planting material production of
phytochemicals and high-value
pharmaceuticals cosmetics and food
additives production of improved and
tailor-made crops through transgenic for
example bt cotton and conservation of
endangered threatened and a rare species
of plants
you
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