भ्रूण बचाव (Embryo Rescue):-
सामान्य परिचय (General Introduction):-
· परिभाषा (Definition):- ऐसा अपरिपक्व या कमजोर भ्रूण जो नष्ट हो सकता है व अपने आप पौधे में विकसित नहीं हो सकता उसे ऊतक संवर्धन तकनीक के द्वारा स्वस्थ पौधे में विकसित करने की प्रक्रिया को भ्रूण बचाव कहते हैं।
(Through tissue culture technique, the process of developing a healthy plant from an immature or weak embryo, which can be destroyed and cannot develops into the plant on its own, is called embryo rescue.)
· दूरस्थ संकरण से भ्रूणपोष का सही प्रकार से विकास नहीं हो पाता है जिससे भ्रूण के नष्ट होने की संभावना बढ़ जाती है।
(Endosperm does not develop properly in distant hybridization, which increases the chances of embryos being destroyed.)
· भ्रूण बचाव प्रक्रिया में कृत्रिम पोषक माध्यम भ्रूणपोष के विकल्प का कार्य करता है। जिससे भ्रूण का विकास बिना रुके लगातार चलता रहता है।
(The artificial nutrient medium serves as an alternate of endosperm in the embryo rescue process. Due to which the development of the embryo continues continuously without stopping.)
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Benefits of In Vitro Pollination:- In vitro pollination is the technique of carrying out pollination under sterile laboratory conditions on cultured ovules, ovaries, or placentas. It helps overcome barriers that prevent normal fertilization.
Benefits:-
> Overcomes pre-fertilization barriers by bypassing stigma and style incompatibility.
> Facilitates wide hybridization between distantly related species or genera.
> Produces hybrids that cannot be obtained through conventional pollination.
> Useful in self-incompatible plants, allowing successful fertilization.
> Improves seed set in plants with poor natural pollination.
> Reduces pollen competition, ensuring desired pollen fertilizes the ovule.
> Helps rescue rare or endangered species by enabling controlled fertilization.
> Supports embryo rescue, reducing embryo abortion after fertilization.
> Useful in plant breeding for transferring desirable traits such as disease resistance and stress tolerance.
> Saves time and space, as pollination and fertilization occur under controlled laboratory conditions.
> Allows study of pollen germination and pollen tube growth under controlled conditions.
> Produces disease-free and genetically valuable progeny when combined with tissue culture techniques.
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साइब्रिड्स (Cybrids):- जब कायिक संकरण के दौरान एक जाति से केन्द्रक व कोशिकाद्रव्य तथा दूसरी जाति से केवल कोशिकाद्रव्य आते हैं तो इस प्रकार बने संकर को साइब्रिड कहते हैं।
(When nucleic and cytoplasm from one species and only cytoplasm from another species come during somatic hybrids, the hybrids thus formed are called cybrids.)
Why Male Sterility is Exploited for Crop Improvement:-
> Eliminates the need for manual emasculation.
> Facilitates economical hybrid seed production.
> Ensures cross-pollination and genetic purity.
> Increases hybrid vigour (heterosis).
> Reduces labour and production costs.
> Improves seed yield and quality.
> Enables large-scale commercial hybrid seed production.
Role of Enzymes in Protoplast Isolation:-
Cellulase:- Digests cellulose of the cell wall.
Pectinase (Macerozyme):- Dissolves the pectin-rich middle lamella.
Hemicellulase:- Breaks down hemicellulose.
Facilitates protoplast release:- Frees intact protoplasts from plant tissues.
Maintains viability:- Gentle enzymatic digestion preserves living, functional protoplasts.
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कायिक क्लोनीय विविधताएँ (Somaclonal Variations):-
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 व संरचना में परिवर्तन हो जाता है जिससे पादपों के निम्न लक्षणों में परिवर्तन आ जाता है:-
(In this, the number and structure of chromosomes changes, due to which the following characteristics of plants change.)
i. पर्ण की आकृति व 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.)
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Scope of Rare Hybrids in Crop Improvement:- Rare hybrids are hybrids produced from distantly related species, genera, or unusual parental combinations that are difficult to obtain through conventional breeding. They serve as valuable sources of novel genetic variation and have great potential in crop improvement.
Scope of Rare Hybrids:-
i. Broadening Genetic Base:-
> Introduce new genes from wild relatives and distant species.
> Reduce genetic vulnerability of cultivated crops.
ii. Transfer of Disease Resistance:-
> Introduce resistance against fungal, bacterial, viral, and nematode diseases.
> Example: Transfer of rust resistance from wild wheat relatives.
iii. Insect and Pest Resistance:-
> Incorporate genes for resistance to insect pests.
> Reduces dependence on chemical pesticides.
iv. Abiotic Stress Tolerance:-
> Develop varieties tolerant to drought, salinity, heat, cold, and flooding.
> Improves crop performance under climate change.
v. Improvement of Yield:-
> Exploitation of hybrid vigour (heterosis).
> Increase biomass and grain yield.
vi. Quality Improvement:- Enhance nutritional quality, protein content, vitamins, oil quality, and processing characteristics.
vii. Development of New Crop Types:-
> Produce synthetic species, amphidiploids, and bridge hybrids.
> Create crops with desirable combinations of traits.
viii. Transfer of Cytoplasmic Traits:- Introduce cytoplasmic male sterility (CMS) for hybrid seed production.
ix. Evolutionary and Genetic Studies:- Help understand chromosome pairing, gene transfer, genome evolution, and speciation.
x. Pre-breeding Material:- Rare hybrids serve as a valuable source for future breeding programmes and gene introgression.
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कायिक भ्रूणजनन (Somatic Embryogenesis):-
1. सामान्य परिचय (General Introduction):-
· कायिक भ्रूणजनन (Somatic Embryogenesis):- कायिक कोशिका से कायिक भ्रूण के निर्माण की प्रक्रिया को कायिक भ्रूणजनन कहते हैं।
(The process of the development of a somatic embryo from a somatic cell is called somatic embryogenesis.)
· भ्रूणाभ (Embryoid):- जाइगोटिक भ्रूण की तुलना में कायिक भ्रूण छोटा व सुसंगठित संरचना होता है जिसे भ्रूणाभ कहते हैं।
(In comparison to the zygotic embryo, the somatic embryo is a small and well-organized structure called the embryoid.)
2. इतिहास (History):-
· J. Reinert (1958-59):- इसने सबसे पहले गाजर में कृत्रिम कायिक भ्रूणजनन को देखा।
(He first observed artificial somatic embryogenesis in carrot.)
· F. C. Steward, M. O. Mapes and K. Mears (1958):-
इन्होने गाजर में स्वतंत्र निलंबित कोशिकाओं से कायिक भ्रूणजनन को देखा। इन्होने कायिक भ्रूणजनन में नारियल पानी(Coconut milk) के महत्व को बताया।
(They observed somatic embryogenesis from freely suspended cells in carrot. He explained the importance of coconut water in somatic embryogenesis.)
· N. S. Rangaswamy (1961):- इसने नींबू में कायिक भ्रूणजनन का विस्तार से अध्ययन किया।
(He studied the somatic embryogenesis in lemon in detail.)
· R. N. Konar and K. Nataraja (1969):- इन्होने Ranunculus sceleratus के विभिन्न पुष्पीय भागों व कायिक ऊतकों को लेकर कायिक भ्रूणजनन का अध्ययन किया।
(He studied somatic embryogenesis by taking different floral parts and somatic tissues of Ranunculus sceleratus.)
· P. V. Ammirato (1974):- इसने Carum carvi की कोशिकाओं से विकसित हो रहे कायिक भ्रूणो पर ऐब्सिसिक अम्ल के प्रभाव का अध्ययन किया।
(He studied the effect of abscisic acid on somatic embryos developing from Carum carvi cells.)
3. सिद्धान्त (Principle):-
· जाइगोटिक भ्रूण व कायिक भ्रूण दोनों परिवर्धन का समान पैटर्न प्रदर्शित करते हैं।
(Both zygotic embryos and somatic embryos exhibit similar patterns of development.)
· एकबीजपत्री पादपों में दोनों प्रकार के भ्रूण आदर्श रूप से निम्न विकासीय अवस्थाओं से होकर गुजरते हैं –
(In monocot plants both types of embryos typically pass through the following developmental stages -)
i. ग्लोबुलर अवस्था (Globular Stage)
ii. स्कुटैलर अवस्था (Scutellar Stage)
iii. कोलिओप्टाइलर अवस्था (Coleoptilar Stage)
· द्विबीजपत्री व कोनिफर पादपों में दोनों प्रकार के भ्रूण आदर्श रूप से निम्न विकासीय अवस्थाओं से होकर गुजरते हैं –
(In dicot and conifer plants, both types of embryos typically pass through the following developmental stages -)
i. ग्लोबुलर अवस्था (Globular Stage)
ii. हिरद्य अवस्था (Heart Stage)
iii. टॉरपीडो अवस्था (Torpedo Stage)
iv. बीजपत्रीय अवस्था (Cotyledonary Stage)
· भ्रूण विकास द्विध्रुवीय होता है। एक सिरे पर प्ररोह व विपरीत सिरे पर मूल निर्माण के लिए ऊतक होता है।
(Embryo development is bipolar. There is one end for shoot formation and the opposite end for root formation.)
· प्रारम्भिक कोशिका में एक असमान विभाजन होता है जिससे एक छोटी व एक बड़ी कोशिका बनती है। बड़ी कोशिका रिक्तिकामय होती है। छोटी कोशिका सघन कोशिकाद्रव्य युक्त होती है जिसे भ्रूणजनित कोशिका कहते हैं।
(The initial cell undergo an unequal cell division, forming a small and a large cell. A large cell is vacuolated. The small cell contains dense cytoplasm called embryonic cells.)
· अब भ्रूणजनित कोशिका 2 प्रकार से विभाजन कर सकती है-
(Now embryonic cells can divide in 2 ways-)
i. अनियमित विभाजनों द्वारा कैलस ऊतक का निर्माण कर सकती है।
(Callus tissue can be formed by irregular cell divisions.)
ii. नियमित व अधिक सुसंगठित विभाजनों द्वारा कायिक भ्रूण का निर्माण कर सकती है।
(By regular and more organized cell divisions, the somatic embryo can form.)
· बाद में मूल शिखाग्र व प्ररोह शिखाग्र बन जाते हैं और ग्लोबुलर अवस्था विकसित हो जाती है। इसके पश्चात अन्य अवस्थाओं से होता हुआ भ्रूण परिपक्वता की ओर बढ़ता है।
(Later the root apex and shoot apex are formed and the globular stage develops. After this, the embryo goes towards maturity through other stages.)
· जाइगोटिक भ्रूणजनन में भ्रूण परिपक्व हो जाता है और निम्न लक्षण प्रदर्शित करता है –
(In zygotic embryogenesis, the embryo matures and exhibits the following characteristics -)
i. परिपक्व भ्रूण आकारिकी
(Mature Embryo Morphology)
ii. संग्रहित कार्बोहाइड्रेट्स, लिपिड्स व प्रोटीन्स का जमाव
(Accumulation of storage carbohydrates, lipids and proteins)
iii. जल की मात्रा में कमी
(Reduction in water content)
iv. उपापचयन में क्रमिक कमी
(Gradual decline of metabolism)
· सामान्यतया कायिक भ्रूण पूर्ण रूप से परिपक्व नहीं होता है। इसकी बजाय वातावरणीय कारकों के कारण कायिक भ्रूण अपने सामान्य विकासीय पैटर्न से विचलित हो जाता है जिसके 3 भविष्य हो सकते हैं –
(Normally the somatic embryo is not fully mature. Instead, due to environmental factors, the somatic embryo deviates from its normal developmental pattern, which can have 3 fates -)
i. कैलस उत्पन्न कर सकता है।
(Callus can be produced.)
ii. प्रत्यक्ष द्वितीयक भ्रूणजनन कर सकता है।
(May perform direct secondary embryogenesis.)
iii. असामयिक रूप से अंकुरित हो सकता है।
(May germinate before maturation.)
· कायिक भ्रूणजनन 2 प्रकार से हो सकता है–
(There are two types of somatic embryogenesis-)
a. प्रत्यक्ष भ्रूणजनन (Direct Embryogenesis)
b. अप्रत्यक्ष भ्रूणजनन (Indirect Embryogenesis)
a. प्रत्यक्ष भ्रूणजनन (Direct Embryogenesis):- जब कर्तोतक की कोशिकाएं कैलस न बनाकर सीधे भ्रूण में परिवर्धित हो जाती हैं तो इसे प्रत्यक्ष भ्रूणजनन कहते हैं। यह बहुत कम देखने को मिलता है। उदाहरण – ओरचार्ड घास (Dactylis glomerata) के संवर्धन में पर्ण मध्योतक कोशिकाओं से सीधे कायिक भ्रूणो का निर्माण होता है।
(When the cells of the explant do not form a callus and develop directly into an embryo, it is called direct embryogenesis. This is rarely seen. Example - In culturing of Orchard grass (Dactylis glomerata), somatic embryos are produced directly from the mesophyll cells.)
b. अप्रत्यक्ष भ्रूणजनन (Indirect Embryogenesis):- जब कर्तोतक की कोशिकाएं पहले कैलस में विकसित होती हैं और फिर कैलस की किसी एक भ्रूणजनित कोशिका से कायिक भ्रूण परिवर्धित होता है तो इसे अप्रत्यक्ष भ्रूणजनन कहते हैं। यह बहुत अधिक देखने को मिलता है।
(When the explant cells first develop into a callus and then the somatic embryo develops from one of the embryonic cell of the callus, it is called indirect embryogenesis. It is most commonly seen.)
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साइब्रिड्स (Cybrids):- जब कायिक संकरण के दौरान एक जाति से केन्द्रक व कोशिकाद्रव्य तथा दूसरी जाति से केवल कोशिकाद्रव्य आते हैं तो इस प्रकार बने संकर को साइब्रिड कहते हैं।
(When nucleic and cytoplasm from one species and only cytoplasm from another species come during somatic hybrids, the hybrids thus formed are called cybrids.)
Applications of Cybrids (साइब्रिड्स के अनुप्रयोग):-
i. Cybrids are important for the transfer of Cytoplasmic male Sterility, antibiotic and herbicide resistance in agriculturally useful plants.
(साइब्रिड्स कृषि उपयोगी पौधों में कोशिकाद्रव्यी नर बाँझपन, एंटीबायोटिक और शाकनाशी प्रतिरोध के हस्तांतरण के लिए महत्वपूर्ण हैं।)
ii. Two different parental genome that can not reproduce sexually (a sexual or sterile) are recombined.
[दो अलग-अलग पैतृक जीनोम जो लैंगिक रूप से प्रजनन नहीं कर सकते (लैंगिक या बंध्य) पुनर्संयोजित होते हैं।]
iii. Overcomes sexual incompatibility barriers.
(लैंगिक असंगति की बाधाओं को दूर करता है।)
iv. Used in the study of cytoplasmic genes and their activities plant breeding experiments.
(कोशिकाद्रव्यी जीन और उनकी गतिविधियों के अध्ययन में पादप प्रजनन प्रयोगों में उपयोग किया जाता है।)
v. Used in mitochondrial research and have been used to provide suggestive evidence of mitochondrial involvement in Alzheimer’s disease, Parkinson’s disease.
(माइटोकॉन्ड्रियल अनुसंधान में उपयोग किया जाता है और अल्जाइमर रोग, पार्किंसंस रोग में माइटोकॉन्ड्रियल की भागीदारी के सुझावात्मक प्रमाण प्रदान करने के लिए उपयोग किया गया है।)
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Role of Mutants in Crop Improvement:- Mutants are plants that possess heritable changes (mutations) in their genetic material (DNA). These mutations may occur naturally (spontaneous mutations) or be induced artificially using physical mutagens (e.g., gamma rays, X-rays) or chemical mutagens (e.g., EMS, sodium azide). Mutation breeding is an important approach for creating genetic variability and developing improved crop varieties.
Role of Mutants in Crop Improvement:-
1. Development of High-Yielding Varieties:-
> Mutants with increased grain number, larger seeds, or enhanced biomass are selected.
> Such mutants contribute to higher crop productivity.
Example:- High-yielding rice and wheat mutant lines developed through mutation breeding.
2. Improvement of Disease Resistance:-
> Mutations can produce resistance genes against fungal, bacterial, and viral diseases.
> Resistant varieties reduce dependence on chemical pesticides.
Examples:-
i. Disease-resistant barley mutants.
ii. Blast-resistant rice mutants.
3. Development of Pest Resistance:-
> Mutants with thicker cuticles, leaf hairs (trichomes), or biochemical resistance can tolerate insect attack.
> Reduces crop losses and pesticide use.
4. Abiotic Stress Tolerance:-
> Mutants have been developed for resistance to:
i. Drought
ii. Salinity
iii. Heat
iv. Cold
v. Flooding
> These traits help maintain productivity under adverse environmental conditions.
5. Improvement of Quality Traits:- Mutations improve:
> Protein content
> Oil content
> Vitamin content
> Mineral composition
> Cooking and processing quality
Examples:-
i. High-lysine maize mutants.
ii. Low-erucic acid rapeseed lines.
6. Early Maturity:-
> Early flowering and early maturing mutants escape drought, frost, and terminal heat stress.
> Enable multiple cropping systems.
7. Semi-Dwarf and Lodging Resistance:-
> Semi-dwarf mutants possess stronger stems and reduced plant height.
> These varieties respond well to fertilizers and have higher yields.
Examples:- Semi-dwarf rice and wheat varieties.
8. Improvement in Plant Architecture:-
> Mutants help develop:
i. Compact plants
ii. Erect leaves
iii. Better branching
iv. Uniform flowering
v. Determinate growth habit
> These characteristics facilitate mechanized cultivation and harvesting.
9. Herbicide Resistance:-
> Herbicide-resistant mutants allow effective weed control without damaging the crop.
> Useful in conservation agriculture.
10. Seedless and Male-Sterile Mutants:-
> Male-sterile mutants are extensively used in hybrid seed production.
> Seedless mutants are desirable in fruit crops.
11. Breaking Undesirable Linkages:-
> Mutation can eliminate undesirable genes while retaining desirable traits.
> Useful when conventional breeding is difficult.
12. Broadening the Genetic Base:-
> Mutation introduces new genetic variation where natural variability is limited.
> Provides novel alleles for plant breeding.
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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 व 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.)
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Use of Biotechnology for Biodiversity Conservation:- Biotechnology plays a vital role in the conservation of biodiversity by protecting endangered species, preserving genetic resources, restoring degraded ecosystems, and supporting sustainable utilization of biological resources. It combines molecular biology, genetics, tissue culture, and bioinformatics to develop effective conservation strategies.
1. Conservation of Endangered Species:- Biotechnology helps conserve rare and endangered plant and animal species through tissue culture, micropropagation, and cryopreservation. These techniques enable rapid multiplication and long-term storage of valuable genetic material.
2. Gene Banks and Cryopreservation:- Seeds, pollen, embryos, DNA, and tissues can be preserved in gene banks or stored at ultra-low temperatures using cryopreservation. This prevents the loss of genetic diversity and provides material for future breeding and restoration programs.
3. DNA Fingerprinting and Molecular Markers:- Molecular markers such as RAPD, AFLP, SSR, and SNPs are used to assess genetic diversity, identify species, detect hybrids, and monitor populations. This information is essential for planning conservation strategies.
4. In Vitro Conservation:- Plant tissue culture techniques allow the conservation of species that produce recalcitrant seeds or are difficult to propagate naturally. In vitro conservation requires less space and protects plants from diseases and environmental hazards.
5. Assisted Reproduction:- Biotechnological methods such as artificial insemination, embryo transfer, and in vitro fertilization (IVF) help increase populations of endangered animal species.
6. Genetic Engineering:- Genetic engineering can develop disease-resistant and stress-tolerant plants, reducing pressure on wild populations and helping restore threatened ecosystems.
7. Environmental DNA (eDNA):- Environmental DNA analysis allows scientists to detect species from water, soil, or air samples without disturbing them. It is useful for monitoring biodiversity and detecting invasive species.
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Use of somatic embryogenesis in crop improvement:-1. Large-scale propagation:-
> Depending on the plant species, virtually unlimited numbers of embryos can be generated from a single explant.
> Drew (1980) estimated that one liter of a carrot suspension culturecontained 1.35 million somatic embryos. Thus, in comparison toconventional propagation methods (e.g., rooted cutting, grafting)
and other in vitro regeneration method (e.g., organogenesis), somatic embryos offer better potential for large-scale propagationsystems.
> Even greater economies of scale may be possible if bioreactor and continuous culture technologies can be applied to embryogenic systems.
> Production of synthetic seeds by encapsulating somatic embryos, shoot buds or any other meristmatic tissue.
> Scope of Synthetic Seeds:-
i. Propagation:-
- Rare and Endangered Plants.
- Elite Genotype.
- Genetically Engineered Plants.
- Seedless Plants.
- Commercially important plants.
ii. Conservation:- Long and Short Term Storage.
iii. Transport:-
- Exchange of Axenic Plant.
- Material Free of Undesirable Contaminants.
2. In vitro selection:-
> In vitro culture of plant cells, tissues or organs on a medium containing selective agents offers the opportunity to select and regenerate plants with desirable characteristics. The technique has also been effectively utilized to induce tolerance which includes the use of some selectiveagents that permit the preferential survival and growth of desired phenotypes
> The presence of a selective agent in the embryogenesis induction or callus initiation medium could increase the probability of recovering stress tolerant plants through somatic embryogenesis.
i. NaCl = Salt tolerant
ii. PEG or mannitol = Drought tolerant
iii. Phytotoxin i.e. fusaric acid or culture filtrate or Pathogen it self = Disease resistant
3. Genetic transformation:- Due to its potentially high multiplication rates andpotential for scale-up via bioreactor, somatic embryogenesis has been emphasized as a suitabletarget for gene transfer.
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Isolation of Mutants:-
Introduction:-
> Mutation occurring in microorganism can be detected and efficiently isolated from the parent organism of other mutants.
> While studying we must be aware of wild type characters of an organism ,so the mutants can easily detected.
> In bacteria and other haploid microorganism, the detection system are straight forward because any new allele should be observed immediately.
> In albino mutation, the detection is very simple. It requires only change in colour of bacterial colony. > The other detection systems are rather complex.
Some Detection Methods:-
1.Replica plating technique
2. Resistance selection method
3. Substrate utilization method
4. Ames method
1.Replica plating technique:-
> Joshua and Esther Ledgerberg (1952) developed a new technique called replica plating .
> This technique is used to detect auxotrophic mutants and wild type strains on the basis of ability to grow in the absence of amino acids.
> Also this test is used to demonstrate the presence of antibiotic resistance in bacterial cultures prior to exposure of antibiotic
> Steps Involved:-
i. Generate the mutants by treating a culture with a mutagen e.g.nitrosoguanidine .
ii. Inoculate a plate containing complete growth medium and incubate it at proper temperature. Both wild type and mutant survivors will from complete medium.
iii. This plate containing complete medium is called master plate.
iv. Prepare a piece of sterile velvet and gently on the upper surface of the master plate to pick up bacterial cell from each colony.
v. As pressed the master plate, again gently press the velvet on the replica plates containing complete medium in one set and lacking cine in only leucine in the other set.
vi. Thus, the bacterial cells are transferred in replica plates in the same position as in master plate.
vii. Incubate the plates and compare the replica plate with master plate for bacterial colony not growing on replica plate.
2. Resistance selection method:-
> This is another method used for isolation of mutants.
> Generally the wild the wild type cells not resistant either to antibiotics or bacteriophage.
> Therefore, it is possible to grow the bacterium in the presence of agent.
> This method is applied for isolation of mutants resistant to chemical compounds that can be amended in agar, phage resistant mutants.
3. Substrate utilization method:-
> This method is employed in the selection of bacteria. Several bacteria utilize only a few carbon sources.
> The cultures are plated on to medium containing alternate carbon sources.
> Any colony that grows on medium can use the substrate and are possibly mutants. These can be isolated.
> Sugar utilization mutants are also isolated by means of color indicator plates.
> EMB medium is used for this purpose.
> This medium contain lactose sugar as carbon source and complete mixture of amino acids.
> Therefore both lactose wild type and lactose mutant cells can grow and form colonies on EMB agar plates.
> The lac+ cells catabolize lactose and secrete acids,therefore the pH of the medium decreases. This will result in staining of colony to dark purple.
> On the other hand, Lac- cells are unable to utilize lactose and use some of the amino acids as carbon source.
> After utilization of amino acid, ammonia is produced that increases the pH and de colorize the dye resulting in white colony.
4. Ames method:-
> Ames test In 1974 Bruce Ames developed a method for evaluating the potential of chemical to cause cancer, known as Ames test .
> Ames test is based on the principle that both cancer and mutations results from the damage of DNA, and results of experiments have demonstrated that 90% of known carcinogen are also mutagens.
> Several species of salmonella typhimurium are employed. Each strain contains a different mutation in the operon histidine biosynthesis.
> Steps:-
i. Prepare the culture of Salmonella histidine auxotrophs (His-).
ii. Mix the bacterial cells and test substance( mutagen) in dilute molten top agar with a small amount of histidine in one set, and control with cmplete medium plus large amount of histidine.
iii. Pour the molten mix on the top of minimal agar plates and incubate at 37°C for 2-3 days.
iv. Until histidine is depleted all the His- cells will grow in the presence of test mutagen.
v. When the histidine is completely exhausted only the revertants will grow on the plate.
vi. The number of spontaneous revertants is low, whereas the number of revertant induced by carcinogen is quite high.
vii. High number of colonies represent the greater mutagenicity.
viii. A mammalian liver extract is added to the above molten top agar before plating.
ix. The extract converts the carcinogen in to electrophilic derivatives which will soon react with DNA molecule.
x. In natural way it is occurs in mammalian system when foreign particle are metabolized in the liver.
xi. Bacteria does not have metabolizing capacity, therefore, the liver extract is added to this test, to promote transformation.
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Role of Biotechnology in Horticulture and Forestry:- Biotechnology has become an essential tool in the improvement of horticultural and forestry crops by enhancing productivity, quality, disease resistance, and environmental sustainability. It integrates techniques such as plant tissue culture, genetic engineering, molecular markers, genome editing, and molecular diagnostics.
Role in Horticulture:-
> Micropropagation enables rapid multiplication of disease-free, uniform planting material of banana, potato, orchids, strawberry, and ornamental plants.
> Genetic engineering develops crops resistant to insects, diseases, herbicides, and abiotic stresses such as drought and salinity.
> Quality improvement includes enhancement of fruit size, color, flavor, nutritional value, and shelf life. For example, delayed fruit ripening reduces post-harvest losses.
> Marker-assisted selection (MAS) accelerates breeding by identifying desirable genes at the seedling stage.
> Production of virus-free plants through meristem culture significantly improves crop yield and quality.
> Synthetic seeds and cryopreservation facilitate germplasm conservation and exchange.
Role in Forestry:-
> Clonal propagation through tissue culture enables mass multiplication of elite tree species such as eucalyptus, teak, bamboo, and poplar.
> Genetic transformation improves wood quality, growth rate, disease resistance, and tolerance to drought and salinity.
> Conservation biotechnology helps preserve endangered forest species through in vitro conservation and cryopreservation.
> Molecular markers assist in genetic diversity analysis, species identification, and breeding programs.
> DNA fingerprinting is useful for tree improvement, certification of planting material, and protection of plant varieties.
> Biotechnology also supports phytoremediation, where genetically improved plants help remove pollutants from contaminated soils.
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