Lecture - 13 Protoplast culture: Isolation, purification and culture, protoplast fusion, Cybrids, Somatic hybrids and Somaclonal variations

Protoplast culture: Isolation, purification and culture, protoplast fusion, Cybrids, Somatic hybrids and Somaclonal variations:-
Somatic Hybridization:- The process of obtaining hybrid plants by fusion of protoplasts of two different species or varieties, is called somatic hybridization. It has 4 main steps -
1. Protoplast Isolation
2. Protoplast Fusion
3. Selection of Hybrid Cells
4. Culturing of Hybrid Cells
1. Protoplast Isolation:- It has 2 main steps -
a. Sterilization of Leaf
b. Enzyme Treatment
a. Sterilization of Leaf:- Soak the leaf in 70% ethanol for 1 minute. After this, keep this leaf in 2% NaOCl solution for 20 to 30 minutes. Now wash this leaf with distilled water for 3 minutes.
b. Enzyme Treatment:- Now this sterilized leaf is treated with 2 enzymes, pectinase and cellulase, respectively. The pectinase enzyme disintegrates the middle lamella. Cellulase enzymes decompose the cell wall. As a result protoplasts are obtained. To increase osmotic concentration, add 500 - 800 ml / L of sorbitol or mannitol.
2. Protoplast Fusion:- For this, 2 different protoplast samples are mixed together. Now Fusogen is added to this mixture which induces protoplast fusion.
One of the following factors can be used as Fusogen -
i. PEG (Poly Ethylene Glycol)
ii. High pH + Ca2+
iii. NaNO3
iv. Electric Pulse
At the end of this step, 3 types of products are obtained -
i. Homokaryon
ii. Heterokaryon
iii. Unfused protoplasts
3. Selection of Hybrid Cells:- It has several methods -
i. Visual markers
ii. Staining of protoplast with fluorescent dyes
iii. Culture, Regeneration and Identification
iv. Red anthocynin marker and green protoplast marker:-
Tobacco (green)  X   Carrot (red)
v. Selective markers:-
Grape   X    Soybean
4. Culturing of Hybrid Cells:-
Ø  Isolated hybrid protoplast cells are proliferated on solid MS-medium in patridishes.
Ø  Osmaticum:- It is a substance that acts to increase the osmotic concentration. It is added to both enzyme mixture and culture medium. The protoplast requires osmotic protection during isolation and culture because the cell wall has not yet been formed. One of the following can be used as osmaticum -
i. Sorbitol
ii. Manitol
iii. Glucose
iv. Sucrose
Ø  Cover the patridish with a lid and seal it with paraffin wax so that the medium does not dry at 40ºC.
Ø  Now transfer this patridish to the incubator where the temperature is 25 - 28ºC and the light intensity is 2300 lux.
Ø  Cell wall formation starts after a few hours. The first cell division occurs after 2–7 days.
Ø  Multicellular groups are formed after 2–3 weeks.
Ø  Now these cellular groups are transferred to a medium free of osmaticum so that callus or embryo can develop.
Ø  Further differentiation leads to organogenesis and development of hybrid plants.

Types of Hybrids:- There are 3 types of cellular hybrids -
1. Symmetric Hybrids
2. Asymmetric Hybrids
3. Cybrids
1. Symmetric Hybrids:- When the genomes of both species are kept in the cells during mitosis of the hybrid cell, the plants thus developed are called symmetric hybrids.
Example:- Pomato (Potato X Tomato)
2. Asymmetric Hybrids:- When the genome of a single species is extinct from cells during mitosis, the plants thus developed are called asymmetric hybrids.
Example:- Tobacco X Carrot
3. Cybrids:- When nucleic and cytoplasm from one species and only cytoplasm from another species come during somatic hybrids, the hybrids thus formed are called cybrids.


कायिक क्लोनीय विविधताएँ (Somaclonal Variations):-

1. सामान्य परिचय (General Introduction):-

·  परिभाषा (Definition):- पादप ऊत्तक संवर्धन के द्वारा पादपों में उत्पन्न होने वाली आनुवंशिक विविधताएँ जिन्हें लक्षण प्रारूप के रूप में देखा जा सकता है।

(The genetic variations that develops in plants through plant tissue culture and which can be seen as phenotypes.)

·  इसमें गुणसूत्रों की संख्या  संरचना में परिवर्तन हो जाता है जिससे पादपों के निम्न लक्षणों में परिवर्तन  जाता है:- 

(In this, the number and structure of chromosomes changes, due to which the following characteristics of plants change.)

i. पर्ण की आकृति  रंग (Leaf shape and colour)

ii. वृद्धि दर (Growth rate)

iii. स्वभाव (Habit)

iv. लैंगिक उर्वरता (Sexual fertility)

·  ये आनुवंशिक उत्परिवर्तन होते हैं जो पादपों में पीढ़ी दर पीढ़ी वंशागत होते हैं।

(These are genetic mutations that are inherited from generation to generation in plants.)

2. प्रकार (Types):- 2 प्रकार हैं (2 types are)–

a. आनुवंशिक विविधताएँ (Genetic Variations):-

Ø  इन्हें वंशागत विविधताएँ भी कहते हैं।

(They are also called as heritable variations.)

Ø  ये कर्तोंत्तक की कायिक कोशिकाओं में पहले से ही विध्यमान विविधताएँ होती हैं।

(These are already existing variations in the somatic cells of explant.)

Ø  ये उत्परिवर्तनों  अन्य DNA परिवर्तनों के कारण उत्पन्न होती हैं।

(They are caused by mutations and other DNA changes.)

Ø  इनकी आवृति उच्च होती है अर्थात बहुत अधिक देखने को मिलती है।

(Their frequency is high, that is, they are seen very commonly.)

b. अधिआनुवंशिक विविधताएँ (Epigenetic Variations):-

Ø  इन्हें अवंशागत विविधताएँ भी कहते हैं।

(They are also called as non-heritable variations.)

Ø  ये पादप ऊत्तक संवर्धन के दौरान उत्पन्न होने वाली विविधताएँ होती हैं।

(These are the variations that occur during plant tissue culture.)

Ø  ये लक्षण प्रारूप में अस्थायी परिवर्तन के कारण उत्पन्न होती हैं।

(These variations arise due to temporary changes in phenotype.)

Ø  इनकी आवृति कम होती है अर्थात बहुत कम देखने को मिलती हैं।

(Their frequency is low which means they are rarely seen.)

3. कारण (Reasons):- 3 कारण हैं (3 causes are)–

a. कार्यिकीय कारण (Physiological causes)

b. आनुवंशिक कारण (Genetic causes)

c. जैवरासायनिक कारण (Biochemical causes)

a. कार्यिकीय कारण (Physiological causes):-

Ø  जब पादप वृद्धि नियामक संवर्धन के सम्पर्क में आते हैं तो परिवर्तन उत्पन्न करते हैं।

(When plant growth regulators come into contact with culture, they produce variations.)

Ø  संवर्धन परिस्थितियाँ भी पादपों में परिवर्तन उत्पन्न करती हैं।

(Culturing conditions also cause variations in plants.)

b. आनुवंशिक कारण (Genetic causes):-

i. गुणसूत्र संख्या में परिवर्तन से (Change in number of chromosomes):-

Ø  Aneuploidy:- 1 या 2 गुणसूत्रों में कमी या वृद्धि। 

(Increase or decrease in 1 or 2 chromosomes.)

उदाहरण (Example):- (2n ± 1), (2n ± 2)

Ø  Polyploidy:- सम्पूर्ण जीनोम की संख्या में वृद्धि  

(Increase in number of complete genomes.)

उदाहरण (Example):- 2n = 4x, 6x

ii. गुणसूत्र की संरचना में परिवर्तन से (Change in structure of chromosomes):-

Ø  Deletion

Ø  Inversion

Ø  Duplication

Ø  Translocation

iii. जीन उत्परिवर्तन के कारण (Due to gene mutation):-

Ø  Transition

Ø  Transversion

Ø  Insertion

Ø  Deletion

iv. कोशिकाद्रव्यी जीन उत्परिवर्तन (Cytoplasmic gene mutation):- कोशिकाद्रव्य में माइटोकोंड्रिया  हरितलवक में DNA पाया जाता है। इसमें उत्परिवर्तन होने से भी पादपों के लक्षणों में परिवर्तन हो सकते हैं।

(DNA is found in chloroplast and mitochondria in cytoplasm. Changes in plant characters can also occur due to mutation in this DNA.)

v. Transposons के सक्रियण से (Due to activation of transposons):- जीनोम में कुछ ऐसे DNA खण्ड पाये जाते हैं जो अपनी स्थिति बदल सकते हैं। इन्हें Transposons कहते हैं। ये भी पादपों के लक्षणों में परिवर्तन कर सकते हैं।

(There are some DNA fragments in the genome that can change their position. They are called Transposons. They can also cause changes in plant characters.)

vi. DNA क्षार क्रम (DNA Base Sequence):-

Ø  DNA के क्षार क्रम में परिवर्तन से विविधता उत्पन्न हो सकती है। RE के उपयोग से DNA खंडों के आकार में परिवर्तन का पता कर सकते हैं।

(Variations can arise due to changes in the base sequence of DNA. Changes in the size of DNA fragments can be detected using RE.)

Ø  प्रोटीन में परिवर्तन से विविधता उत्पन्न हो सकती है। इसमें प्रोटीन के साथ खण्ड जुड़ता है या अलग होता है। विशिष्ट प्रोटीन के स्तर में परिवर्तन हो सकता है।

(Variation can arise due to changes in proteins. In this, the fragment is added to protein or separates from the protein. Specific protein levels may change.)

Ø  DNA के मेथाइलीकरण से विविधता उत्पन्न हो सकती है। मेथाइलीकरण से अनुलेखन की प्रक्रिया बन्द हो जाती है।

(Variation can arise due to methylation of DNA. Methylation inhibit the transcription process.)

c. जैवरासायनिक कारण (Biochemical causes):-

Ø  कार्बन मेटाबोलिज्म में परिवर्तन से प्रकाश संश्लेषण की क्षमता खत्म हो सकती है।

(Changes in carbon metabolism can deplete the capacity of photosynthesis.)

Ø  कैरोटिनोइड परिपथ के माध्यम से स्टार्च का जैवसंश्लेषण होने से भी पादपों के लक्षणों में परिवर्तन  सकते हैं।

(Biosynthesis of starch via the carotenoid pathway can also cause changes in plant characters.)

Ø  नाइट्रोजन मेटाबोलिज्म में परिवर्तन से तथा प्रतिजैविक रोधिता से भी पादपों के लक्षणों में परिवर्तन  सकते हैं।

(Changes in nitrogen metabolism and antibiotic resistance can also cause changes in plant characters.)


4. फसल उन्नयन में उपयोग (Use in Crop Improvement):-

a. सस्य रूप से उपयोगी पौधों का उत्पादन (Production of agronomically useful plants):- अनेक फसलों में उत्कृष्ट लक्षण कायिक क्लोनीय विविधता से विकसित हुए हैं। उदाहरण – धान, गेहूँ, मक्का, गन्ना, जौं, जई, सोयाबीन, आलू, टमाटर, गाजर, सरसों, तंबाकू आदि।

(In many crops, superior traits have developed from the somaclonal variations. Examples - Paddy, Wheat, Maize, Sugarcane, Barley, Oats, Soybean, Potatoes, Tomatoes, Carrots, Mustard, Tobacco etc.)

b. रोगों के प्रति रोधिता (Resistance to diseases):-अनेक फसलों में कायिक क्लोनीय विविधता के उपयोग से रोग रोधिता विकसित की गई है। उदाहरण:- धान, गेहूँ, मक्का, गन्ना, जौं, आलू, टमाटर, गाजर, तंबाकू, सेब, केला, एल्फाएल्फा आदि।

(Disease resistance has been developed in many crops using the somaclonal variations. Examples: - Paddy, Wheat, Maize, Sugarcane, Barley, Potatoes, Tomatoes, Carrots, Tobacco, Apples, Bananas, Alphalfa etc.)

c. अजैविक प्रतिबलों से रोधिता (Resistance to abiotic stresses):-

i. हिमीकरण सहिष्णुता (Freezing tolerance):- 

उदाहरण (Example) – गेहूँ (Wheat)

ii. लवण सहिष्णुता (Salt tolerance):- 

उदाहरण (Example) – धान (Paddy), मक्का (Maize), तम्बाकू (Tobacco)

iii. एलुमिनियम सहिष्णुता (Aluminium tolerance):- 

उदाहरण (Example) – गाजर (Carrot), ज्वार (Jowar), टमाटर (Tomato)

d. शाकनाशियों से रोधिता (Resistance to herbicides):-

i. तम्बाकू की Glyphosate, Sulfonylurea  Picloram के प्रति रोधिता

(Resistance of tobacco to glyphosate, sulfonylurea and Picloram)

ii. गाजर की Glyphosate के प्रति रोधिता

(Resistance of carrot to glyphosate)

iii. कमल की 2, 4 – D के प्रति रोधिता

(Resistance of Lotus to 2, 4 - D)

e. बीज गुणवत्ता में सुधार (Improvement in Seed Quality):- Lathyrus sativa बीजों की एक नई किस्म Lathyrus Bio L212 का विकास कायिक क्लोनीय विविधता द्वारा किया गया है। इस किस्म के बीज टॉक्सिन रहित होते हैं।

(A new variety of Lathyrus sativa seeds, Lathyrus Bio L212, has been developed by the somaclonal variations. The seeds of this variety are free of toxins.)