PLANT TISSUE CULTURE CSIR

Summary

This 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

00:00

🌿 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.

05:01

🔬 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.

10:03

🌱 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.

15:07

🌱 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.

20:09

🌱 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.

Keywords

💡Mega diverse

The term 'mega diverse' refers to a country or region that is exceptionally rich in biodiversity, harboring a vast number of species, many of which are endemic. In the context of the video, India is described as a 'mega diverse country' with thousands of rare endemic plants and animal species. This term is crucial as it sets the stage for discussing the importance of conservation efforts in such a biodiverse nation.

💡Endemic species

Endemic species are those that are native and restricted to a certain geographic area. The script mentions that India has many endemic plant species, emphasizing the country's unique biodiversity. The video discusses the threat of extinction these species face due to human activities, highlighting the urgency of conservation.

💡Extinction

Extinction refers to the disappearance of a species so that no living member of that species exists. The video script discusses how many endemic species in India are 'on the verge of extinction' due to anthropogenic activities and population influx, underscoring the critical need for conservation strategies.

💡Conservation scientists

Conservation scientists are professionals who work to protect and preserve biodiversity. The script mentions that these scientists use various techniques to preserve medicinal plants, which are crucial for both ecological balance and human use. The video's theme revolves around their efforts to conserve species through plant tissue culture.

💡Plant tissue culture

Plant tissue culture is a set of techniques used to grow plant cells, tissues, or organs under sterile conditions on a nutrient medium. The video focuses on this technique as a method for preserving plant species and improving crops. It is central to the video's message about modern approaches to conservation and crop improvement.

💡Micropropagation

Micropropagation is a method of plant tissue culture that involves the rapid production of new plantlets from small pieces of plant tissue. The script describes this process as a way to produce clones of plants, which is essential for mass propagation and preservation of endangered species, as well as for creating disease-free planting material.

💡Sterile conditions

Sterile conditions are environments free from living organisms such as bacteria, fungi, or viruses, which are necessary for the successful growth of plant tissue cultures. The video script details the steps to ensure sterile conditions, such as the sterilization of culture media and instruments, which is vital for the health and growth of the cultured plant tissues.

💡Explants

Explants are small pieces of plant tissue, such as leaves, buds, or stems, that are used to initiate a tissue culture. The video explains the preparation of explants, emphasizing the need for them to be collected from healthy, disease-free plants. Explants are a key component in the tissue culture process as they serve as the starting point for new plant growth.

💡Acclimatization

Acclimatization in the context of plant tissue culture refers to the process of gradually adjusting plantlets grown in vitro to the environmental conditions of the outside world. The script describes how plantlets are hardened and subjected to various shocks to prepare them for outdoor conditions. This step is crucial for ensuring the survival of the plants after they are removed from the controlled environment of the culture.

💡Endangered species

Endangered species are those that are at risk of extinction due to various threats. The video script discusses the role of plant tissue culture in conserving endangered, threatened, and rare plant species, which is a critical aspect of biodiversity preservation and ecological balance.

💡Transgenic crops

Transgenic crops are plants that have had their genetic material altered through genetic engineering to exhibit certain desirable traits. The video mentions the production of improved and tailor-made crops through transgenic techniques, such as BT cotton, which is an example of how plant tissue culture can be used to develop crops with enhanced characteristics.

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

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