Plant Tissue Culture PPT | Types, Procedure, Application in Agriculture and More ..| PDF Download - Agrobotany

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Conventional breeding methods are widely used in crop improvement, but plant biotechnology offers a faster and more intensive alternative. An example of this is Bt cotton, which has been improved through biotechnology. Plant tissue culture, involving the in-vitro cultivation of plant tissue in controlled conditions, is a key technique in this field.

Understanding the components of plant tissue culture is crucial:

- Plant: An organism with totipotency.

- Tissue: A group of cells isolated from a plant, known as an explant.

- Culture: A favorable environment promoting growth.

Plant tissue culture involves the production of modified plants using tissue from the parent plant in a controlled environment. The plants regenerated from tissue culture are referred to as the R0 generation, with subsequent sexual generations labeled as R1, R2, and so on.

Types of Plant Tissue culture

A. Callus culture

B. Suspension culture

1. Anther culture

2. Embryo culture 

3. Pollen culture

4. Shoot  & Meristem culture 

5. Ovary culture

6. Protoplast culture

Principles of Plant Tissue Culture

Plant tissue culture is based on three key principles:

1. Totipotency: The ability of a single plant cell to develop into a complete plant.

2. Competency: The capacity of cells to differentiate and undergo morphogenesis.

3. Determinism: The ability of a cell to respond to stimuli that trigger development and morphogenesis.

Procedure of Plant Tissue Culture

1. Preparation of Nutrient Medium: Prepare and sterilize the medium based on plant selection.

2. Selection of Explant: Choose any healthy plant part (e.g., bud, leaf, root, seed).

3. Sterilization of Explants: Use a 1-2% solution of sodium/calcium hypochlorite or 0.1% mercuric chloride for sterilization.

4. Inoculation: Transfer the sterile explant to the nutrient medium under aseptic conditions.

5. Incubation: Incubate cultures at 25 ± 2°C with 50-70% relative humidity and 16-hour photoperiod.

6. Callus Growth: Formation of a callus, a mass of undifferentiated cells with potential for organogenesis.

7. Sub-culturing: Transfer callus to fresh medium after about 28 days to prevent nutrient depletion and toxic metabolite accumulation.

8. Organogenesis: Induce organ formation (shoots and roots) in the callus using hormones in the medium.

9. Direct Regeneration: Regenerate explants directly into plantlets to avoid somaclonal variations.

10. Acclimatization: Transfer rooted plantlets to soil and maintain humidity for 15-30 days to adjust to field conditions.

Importance/Application in Agriculture

- Clonal propagation

- Cryopreservation of germplasm

- Overcoming self-sterility

- Genetic transformation

- Early flowering

- Production of synthetic seeds

- Rapid crop multiplication

- Increased biomass energy

- Reduced dormancy period

- Enhanced secondary metabolite production

- Genetic variability

- Disease-free plants

- Germplasm conservation

- Increased resistance to biotic and abiotic stress

Advantages of Plant Tissue Culture

- Enhances genotypic and phenotypic traits.

- Preserves desirable genes and rescues endangered varieties.

- Reduces genetic variability for uniform crop production.

- Allows off-season cultivation.

Disadvantages of Plant Tissue Culture

- Expensive and requires good infrastructure.

- Requires specialized scientific knowledge.

History of Plant Tissue Culture

1902

✓Gottlieb Haberlandt [Father of PTC]

✓ Gave concept of Totipotency

1910

✓ A. Kossel

✓ Gave the concept of Secondary Metabolite.

1925

✓F. Laibach

✔First used the technique of Embryo Culture.

1928

✔Frits Warmolt Went

✔Introduced Phytohormones.

1952

✓G. Morel and Martin

✔Produced the virus free dahlia through meristem culture.

1958

✓ Reinert

✓Developed the somatic embryo in suspension.

1960

✓E. C. Cocking

✓Gave the method of Protoplast isolation and culture.

1962

✓Murashige and Skoog

✓Developed the Tissue culture medium (MS Medium).

1964

✓Guha and Maheshwari

✓Gave the method of Anther culture and Haploid production.

1981

✓Larkin and Scowcroft

✓ Proposed the term of Somaclonal variation.

1985

✓R. B. Horsh et. al.

✔First transgenic plants were produced via Agrobacterium mediated genetic transformation of Nicotiana tabacum (tobacco) protoplasts.

Learn in Hindi

परम्परागत प्रजनन पद्धतियाँ फसल सुधार में सबसे व्यापक रूप से उपयोग की जाती हैं। लेकिन अब पादप जैव प्रौद्योगिकी एक ऐसी पद्धति के रूप में उभर कर सामने आई है जिसके द्वारा पारंपरिक तरीकों की तुलना में फसल सुधार अधिक गहन और तेजी से हो सकता है। बीटी. कपास, पादप जैव प्रौद्योगिकी द्वारा उन्नत फसल का एक अच्छा उदाहरण है। चूंकि टिश्यू कल्चर और जेनेटिक इंजीनियरिंग से फसल सुधार में कम समय लगता है। इसलिए यह और प्रसिद्ध होती जा रहा है।.

प्लांट टिशू कल्चर नियंत्रित स्थितियों में पौधों की कोशिकाओं की इन-विट्रो खेती है।

अन्य विषयों को शुरू करने से पहले आपको प्लांट टिश्यू कल्चर के घटकों को ठीक से समझ लेना चाहिए।

पौधा - एक जीव जिसमें टोटिपोटेंसी की क्षमता होती है।

ऊतक - पौधों से पृथक कोशिकाओं का एक समूह, जिसे एक्सप्लांट के रूप में जाना जाता है।

कल्चर - एक अनुकूल वातावरण जो जीवन को बढ़ावा देता है।

दूसरे शब्दों में - एक कल्चर में मूल पौधे के ऊतक का उपयोग करके संशोधित पौधे का उत्पादन पंत ऊतक संवर्धन के रूप में जाना जाता है।

यह एक उचित परिभाषा नहीं है (क्यों? पहली और दूसरी परिभाषाओं के बीच अंतर खोजें)। क्योंकि बिना नियंत्रित और अनुकूल परिस्थितियों के एक ऊतक पूरा पौधा नहीं बन सकता।

Principles of Plant Tissue Culture 

प्लांट टिशू कल्चर मुख्य रूप से तीन सिद्धांतों पर निर्भर करता है:

टोटिपोटेंसी:

 यह एकल पादप कोशिका की आनुवंशिक क्षमता है जो पूरे पौधे का उत्पादन करती है।

योग्यता:

 कोशिकाएं विभेदीकरण और रूपजनन की क्षमता को बनाए रखती हैं।

नियतत्ववाद:

एक कोशिका की क्षमता जो एक विकासात्मक प्रक्रिया और मोर्फोजेनेसिस को शुरू करती है।

Procedure of Plant Tissue Culture

कल्चर के विकास में प्रयुक्त सामान्य तकनीक का वर्णन इस प्रकार है:

1. उपयुक्त पोषक माध्यम तैयार करना:

पौधे के चयन के अनुसार, माध्यम तैयार किया जाता है और आटोक्लेव किया जाता है (आटोक्लेव द्वारा निष्फल)

2. अन्वेषक का चयन:

एक स्वस्थ पौधे के किसी भी भाग को काटकर उसका उपयोग किया जाता है। उदाहरण – कली, पत्ती, जड़, बीज आदि।

3. अन्वेषकों की स्टेरलाइजेशन :

एक्सप्लांट्स को सोडियम या कैल्शियम हाइपोक्लोराइट के 1-2% घोल या मर्क्यूरिक क्लोराइड के 0.1% घोल से निष्फल किया जाता है।

4. स्थानांतरण:

स्टेराइल( निष्फल ) एक्सप्लांट को अनुकूल परिस्थितियों में ठोस पोषक माध्यम पर स्थानांतरित किया जाता है।

5. ऊष्मायन:

कल्चर्स को 25 ± 2 डिग्री सेल्सियस और 16 घंटे की दीप्तिकालिता के लिए 50-70% तक की सापेक्ष आर्द्रता पर ऊष्मायन किया जाता है।

6. कैलस ग्रोथ:

अन्वेषक की ऊपरी सतह शिथिल रूप से व्यवस्थित पतली दीवार वाली कोशिकाओं के आकारहीन परत से ढक जाती है। ऊतक की इस परत को कैलस कहा जाता है। यह असामान्य वृद्धि की विशेषता है और इसमें जड़ें और अंकुरण पैदा करने की क्षमता है। (और वह क्षमता योग्यता है)

7. उपकल्चर:

■ जब कैलस कुछ दिनों के लिए बढ़ जाता है (मान लीजिए 28 दिन), तो इसे नए माध्यम से स्थानांतरित करना आवश्यक है। अन्यथा, पोषक तत्वों की कमी, जहरीले मेटाबोलाइट्स का संचय और पानी की कमी हो जाएगी और कैलस की मृत्यु हो जाएगी।

8. ऑर्गेनोजेनेसिस:

माध्यम में रसायनों द्वारा दी गई उत्तेजना के द्वारा कैलस में ऑर्गोजेनेसिस शुरू होता है। ऑर्गोजेनेसिस दो चरणों में होता है, अर्थात् कॉलोजेनेसिस या तना बनना ओर इजोजेनेसिस या जड़़ बनना। दोनों प्रकार के ऑर्गेनोजेनेसिस, माध्यम में मौजूद हार्मोन द्वारा नियंत्रित होते हैं।

9. प्रत्यक्ष पुनर्जनन:

कई पौधों में, कैलस के उप-संवर्धन के परिणामस्वरूप अवांछित वृद्धि (सोमाक्लोनल विविधता) में रूपांतर हो जाता हैं। इससे बचने के लिए एक्सप्लांट्स को प्लांटलेट्स में परिवर्तित करनेेे की कोशिश की जा सकती है। यह संवर्धन मीडिया के हार्मोनल संयोजन को बदलकर कई पौधों की प्रजातियों में प्राप्त किया गया है।

10. अनुकूलन:

अंतिम चरण में, जड़ वाले पौधों को अनुकूलन के अधीन किया जाता है, ताकि वे क्षेत्र की परिस्थितियों में आसानी से समायोजित हो सकें। पौधों को माध्यम से बाहर निकाला जाता है, आगर को हटाने के लिए बहते पानी में अच्छी तरह से धोया जाता है और फिर 24-48 घंटों के लिए कम खनिज नमक माध्यम (LMSM) में रखा जाता है। इन पौधों को फिर 1:1:1 के अनुपात में मिट्टी, रेत और पत्ती के सांचों के आटोक्लेव विसंक्रमित मिश्रण वाले बर्तनों में स्थानांतरित किया जाता है। नमी बनाए रखने के लिए बर्तन को आमतौर पर पारदर्शी पॉलीथिन से ढक दिया जाता है। इसे 15-30 दिनों तक बिना छेड़े रखा जाता है। इस अवस्था में पौधा पूरी तरह से जलवायु के अनुकूल हो जाता है।

Importance/Application of Plant Tissue culture in Agriculture 

क्लोनल प्रचार के लिए

जननद्रव्य के क्रायोसंरक्षण के लिए

स्व बाँझपन पर काबू पाने के लिए

आनुवंशिक परिवर्तन के माध्यम से फसलों को संशोधित करने के लिए।

जल्दी पुष्पण के लिए। 

कृत्रिम बीज के उत्पादन के लिए।

बड़े पैमाने पर फसल का तेजी से गुणन करने के लिए।

बायोमास ऊर्जा बढ़ाने के लिए।

सुषुप्ति काल को कम करने के लिए

माध्यमिक चयापचयों की मात्रा बढ़ाने के लिए।

आनुवंशिक परिवर्तनशीलता लाने के लिए।

रोग मुक्त पौधों का उत्पादन करना।

जनन्द्रव संरक्षण के लिए।

जैविक और अजैविक तनाव के खिलाफ प्रतिरोध क्षमता बढ़ाने के लिए ।

Advantages of Plant tissue culture 

प्लांट टिशू कल्चर का दुनिया भर में उपयोग किया जाता है क्योंकि इसके कई फायदे हैं जिनका उल्लेख नीचे किया गया है

1. फसल की  जीनोटाइप क्षमता और फेनोटाइपिक सुंदरता बढ़ाएं। (आनुवांशिक वृद्धि)

2. वांछनीय जीन संरक्षण में सहायता और आनुवंशिक क्षरण की दर में कमी । साथ ही लुप्तप्राय किस्मों के बचाव में मदद करती है। (आनुवंशिक संरक्षण )

3. परिवर्तनशीलता को कम करने के लिए जो फसलों की समान वृद्धि की ओर ले जाती है। (फसलों का समान उत्पादन )

4. प्लांट टिशू कल्चर की सबसे अच्छी बात बेमौसमी खेती है। फसल को पूरे वर्ष किसी भी समय हुआ जा सकता है

Disadvantages of Plant tissue culture 

1. फसल उगाने के महंगा तरीका है।

2. अच्छे आधारभूत संरचना की जरूरत पढ़ती है।

3. विज्ञान की कम जानकारी रखने वाला सामान्य आदमी इसमें भाग नहीं ले सकता। 

History of Plant Tissue Culture

1902

✓गोटलीब हैबरलैंड्ट [PTC के जनक]

✓टोटिपोटेंसी की अवधारणा दी

1910

✓ए. कोसेल

✓सेकेंडरी मेटाबोलाइट की अवधारणा दी।

1925

✓एफ. लाइबैक

✔सबसे पहले भ्रूण संवर्धन की तकनीक का इस्तेमाल किया।

1928

✔फ्रिट्स वार्मोल्ट वेंट

✔फाइटोहॉर्मोन पेश किए।

1952

✓जी. मोरेल और मार्टिन

✔मेरिस्टेम संवर्धन के माध्यम से वायरस मुक्त डाहलिया का उत्पादन किया।

1958

✓रीनर्ट

✓निलंबन में दैहिक भ्रूण विकसित किया।

1960

✓ई. सी. कॉकिंग

✓प्रोटोप्लास्ट अलगाव और संवर्धन की विधि दी।

1962

✓मुराशिगे और स्कोग

✓ऊतक संवर्धन माध्यम (एमएस माध्यम) विकसित किया।

1964

✓गुहा और माहेश्वरी

✓पराग संवर्धन और अगुणित उत्पादन की विधि दी।

1981

✓लार्किन और स्कोक्रॉफ्ट

✓सोमाक्लोनल भिन्नता शब्द का प्रस्ताव दिया।

1985

✓आर. बी. होर्श एट अल.

✔पहले ट्रांसजेनिक पौधे निकोटियाना टैबैकम (तम्बाकू) प्रोटोप्लास्ट के एग्रोबैक्टीरियम मध्यस्थता वाले आनुवंशिक परिवर्तन के माध्यम से उत्पादित किए गए थे।

What is Plant Tissue Culture?

Convention Breeding methods are the most widely used in crop Improvement. But now Plant biotechnology emerge as a method through which crop improvement can be more intensive and fastly than conventional methods. Bt.cotton, is a good example of an improved crop by plant biotechnology. Since tissue culture and genetic engineering take less time in crop improvement. Hence it's becoming more famous.

"Plant Tissue culture is the in-vitro cultivation of plant tissue in controlled or aseptic conditions."

Or

Plant tissue culture refer the growth of living plant tissue in a suitable culture medium and with invitro conditions.

Before starting other topics you should properly understand The COMPONENTS of Plant Tissue culture. 

Plant - An organism having totipotency ability. 

Tissue - A group of cells, isolated from a Plant Know as Explant. 

Culture - A Favorable environment that promotes life. 

In other words - Production of a modified plant Using the Tissue of the parent plant in a culture is known as Pant tissue culture.  

That is not a proper definition (Why? Find a difference between the first and second definitions). Because without providing controlled and favorable conditions a tissue can not be a whole plant. 

*The Plants regenerated from tissue culture are designed as R0 generation and their successive sexual generation as R1 and R2 and soon. 

Related Posts

How many Types of Plant Tissue culture?

There are several types of plant tissue culture techniques, each with specific applications and methods. Some of them main types are following 

Embryo Culture: Isolating and culturing embryos from seeds or immature plants to grow into new plants.

Callus Culture: Growing plant tissues in an undifferentiated mass (callus) on a culture medium, often used for genetic modification and plant regeneration.

Organ Culture: Culturing plant organs such as roots, stems, or leaves to study organ development or for vegetative propagation.

Cell Suspension Culture: Growing plant cells in a liquid medium to produce a large number of cells, often used for biochemical studies and secondary metabolite production.

Protoplast Culture: Isolating and culturing protoplasts (plant cells without cell walls) for genetic engineering and somatic hybridization.

Anther and Pollen Culture: Culturing anthers or pollen to produce haploid plants, useful in plant breeding and genetic studies

Shoot Tip and Meristem Culture: Culturing shoot tips or meristems to produce virus-free plants and for micropropagation.

What is the Principle of Plant Tissue Culture?

Plant tissue culture mainly depends upon three principles:

TOTIPOTENCY:

Genetic potential of a single plant cell to produce the entire plant.

COMPETENCY:

Cells retain the ability for differentiation and morphogenesis. 

DETERMINISM:

The ability of a cell to respond to the stimulus that initiates a developmental process leading to morphogenesis. 

What is the Procedure of Plant Tissue Culture?

The general technique used in the isolation and growth of culture is described as follows:

1. Preparation of Suitable Nutrient Medium:

As per the selection of plant, the medium is prepared and autoclaved ( sterilized by autoclave).

2. Selection of Explant:

Any excised part of a healthy plant can be used e.g. Bud, leaf, root, seed etc.

3. Sterilization of Explant:

Explants can be sterilized by 1-2% solution of sodium or calcium hypochlorite or 0.1% solution of mercuric chloride. 

4. Inoculation (Transfer):

The sterile explant is inoculated or transferred on solidified nutrient medium under aseptic conditions.

5. Incubation:

Cultures are incubated at 25 ± 2°C and a relative humidity of up to 50-70% for 16 hours of photoperiod.

6. Callus Growth:

The upper surface of the explant gets covered by an amorphous mass of loosely arranged thin-walled cells. This mass of tissue is called a callus. It is characterized by abnormal growth and has the potential to produce roots and shoots. ( And that potential is COMPETENCY).

7. Sub-Culturing:

When the callus has grown for some days (say 28 days), it is essential to subculture it on a fresh medium. Otherwise, nutrient depletion, accumulation of toxic metabolites and paucity of water will occur and leads to the death of the callus.

8. Organogenesis:

Organogenesis starts in the callus in response to the stimulation given by the chemicals in the medium. Organogenesis takes place in two stages, namely callogenesis or shoot initiation and rhizogenesis or root initiation. Both types of organogenesis are controlled by the hormones present in the medium. 

9. Direct Regeneration:

In many plants, sub-culturing of callus results in undesired variations of clones (somaclonal variations). To avoid this, direct regeneration of the explants into plantlets can be tried. This has been achieved in many plant species by altering the hormonal combination of the culture media.

10. Acclimatization:

In the last stage, the rooted plantlets are subjected to acclimatization, so that they can easily adjust to the field conditions. The plantlets are taken out from the medium, washed thoroughly in running water to remove the agar and then put in a low mineral salt medium (LMSM) for 24-48 hours. These plantlets are then transferred to pots containing an autoclave sterilized mixture of clay, sand and leaf moulds in 1:1:1 proportion. The pot is usually covered with transparent polythene to maintain humidity. It is kept undisturbed for 15-30 days. At this stage, the plant becomes fully acclimatized. 

What are Application of Plant Tissue Culture in Agriculture?

• Clonal Propagation: Allows for the mass production of genetically identical plants, ensuring uniformity and quality.
• Cryopreservation of Germplasm: Preserves plant genetic material at ultra-low temperatures, safeguarding biodiversity and enabling future crop restoration.
• Overcoming Self-Sterility: Facilitates the breeding of plants that cannot self-pollinate, enhancing crop yields.
• Genetic Transformation: Enables the introduction of new genes into plants, leading to the development of crops with desirable traits such as pest resistance, improved nutrition, and environmental tolerance.
• Early Flowering: Induces flowering at an earlier stage, accelerating breeding programs and reducing the time required to develop new varieties.
• Production of Synthetic Seeds: Produces artificial seeds from somatic embryos, providing an alternative method for plant propagation and conservation.
• Rapid Multiplication of Crops: Enables the quick production of large numbers of plants, which is particularly useful for meeting commercial demands and restoring plant populations.
• Increase in Biomass Energy: Enhances the production of biomass, contributing to renewable energy resources and sustainable agricultural practices.
• Reduced Dormancy Period: Shortens the dormancy period of seeds and plants, allowing for faster crop cycles and increased productivity.
• Increased Production of Secondary Metabolites: Boosts the production of valuable secondary metabolites, which are used in pharmaceuticals, flavors, fragrances, and other industries.
• Genetic Variability: Promotes genetic diversity within plant populations, leading to the development of new and improved varieties with enhanced traits.
• Production of Disease-Free Plants: Generates plants free from pathogens, ensuring healthier crops and reducing the reliance on chemical treatments.
• Germplasm Conservation: Conserves plant genetic resources, protecting endangered species and preserving genetic diversity for future breeding programs.
• Increased Resistance to Biotic and Abiotic Stress: Develops plants with improved resistance to pests, diseases, and environmental stresses, contributing to sustainable agriculture and food security.

What is the Advantage of Plant Tissue Culture?

Plant tissue cultures are used worldwide due there numerous advantages which are Mentioned below 

1. Increase genotypes abilities and phenotypic beauty of crop. (Genetic Enhancement) 

2. Aid in desirable gene preservation and decrease the rate of genetic erosion. Also, help in the rescue of endangered varieties. (Genetic preservation) 

3. To reduce variability which leads to the uniform growth of crops. (Uniform production of crops) 

4. The best thing about plant tissue culture is off-season cultivation. The crop can be sown at any time throughout the year

Explain the Disadvantages of Plant Tissue Culture?

Plant tissue culture, while offering numerous advantages, also has several disadvantages:

1. High Cost: Setting up and maintaining a tissue culture lab is expensive due to the need for specialized equipment, sterile conditions, and skilled personnel.

2. Technical Expertise: Requires skilled technicians and scientists to perform and manage the cultures effectively.

3. Contamination Risk: High susceptibility to microbial contamination, which can compromise the entire culture and result in loss of valuable material.

4. Genetic Variability: Somaclonal variation, or genetic mutations that occur during tissue culture, can lead to plants that are genetically different from the parent plant, which can be undesirable in some applications.

5. Labor-Intensive: The process is labor-intensive, requiring careful monitoring and regular subculturing to maintain healthy cultures.

6. Physiological Disorders: Plants grown in tissue culture may exhibit abnormalities such as hyperhydricity (water-soaked appearance), poor rooting, or abnormal leaf morphology when transferred to soil.

7. Scalability Issues: Scaling up from laboratory to commercial production can be challenging and may not always be feasible for all plant species.

8. Ethical and Legal Concerns: Some ethical and legal issues arise with the use of genetically modified organisms (GMOs) and intellectual property rights associated with plant varieties.

History of Plant Tissue Culture

1902
✓Gottlieb Haberlandt [Father of PTC]
✓ Gave concept of Totipotency

1910
✓ A. Kossel
✓ Gave the concept of Secondary Metabolite.

1925
✓F. Laibach
✔First used the technique of Embryo Culture.

1928
✔Frits Warmolt Went
✔Introduced Phytohormones.
1952
✓G. Morel and Martin
✔Produced the virus free dahlia through meristem culture.
1958
✓ Reinert
✓Developed the somatic embryo in suspension.
1960
✓E. C. Cocking
✓Gave the method of Protoplast isolation and culture.
1962
✓Murashige and Skoog
✓Developed the Tissue culture medium (MS Medium).
1964
✓Guha and Maheshwari
✓Gave the method of Anther culture and Haploid production.
1981
✓Larkin and Scowcroft
✓ Proposed the term of Somaclonal variation.
1985
✓R. B. Horsh et. al.
✔First transgenic plants were produced via Agrobacterium mediated genetic transformation of Nicotiana tabacum (tobacco) protoplasts.

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Reference

Pundhan Singh. 2016. Objectives Plant biotechnology. Kalyani publishes, New Delhi.

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