eChapters

Introduction

One of the biggest problems emerging for the survival of the people in the future is food security. To feed an anticipated nine billion people by 2050, the globe would need to produce roughly twice as much food as it does today. In order to properly accomplish this aim, food production must be elevated sustainably on currently available arable land while addressing the problems brought on by climate change. Over the past five decades, crop yields have steadily increased due to crop breeding programs and better management practices. The rate of yield improvement has, however, reached a plateau (Grassini et al. 2013). The noncompounding yearly yield growth of 0.9% for wheat at the present time, world output rises by 38%, is quite small, and is unable to fulfill expected demand levels by 2050. Therefore, the most beneficial course of action is to investigate more effective, higher-yielding crops (Ray et al. 2013).

Historically, the genetic improvement of wheat has been accomplished by sexual hybridization between related species, giving rise to a large number of cultivars with excellent agronomic performance and high yields. The main methodology for improving cereal crops is still traditional plant breeding, sometimes in combination with traditional cytogenetic methods (Salina et al. 2015). Cereal crops immediately became the top targets for genetic modification due to the worldwide importance of cereal grains in human diets. Over the past 10 years, wheat genetic technology for processing has advanced quickly.

Tissue culture is a technique used to culture cells, tissues, organs or whole plant under controlled environmental conditions that in vitro condition is known as plant tissue culture. Plant tissue culture exploits the totipotency nature of plant cells, ability to develop in to complete plantlet.

Introduction

Tissue culture refers to the technique of growing cells, tissues, or organs under controlled, artificial conditions. This allows propagation of tissues from a small sample of cells/tissues under sterile conditions. Plant tissue culture is widely used for various purposes, including the production of disease-free plants, rapid multiplication of plants with desirable traits, genetic modification, conservation of rare or endangered species, etc. Tissue culture is a valuable technique because it enables consistent and large-scale production of plants, ensures genetic uniformity, and allows manipulation of plant biology at cellular level. In this chapter, we will discuss about the major types of tissue culture techniques and their advantages, disadvantages, as well as applications in brief.

Different Types of Tissue Culture

Callus culture involves induction and growing of unorganized, undifferentiated cell masses from an explant on a nutrient/culture medium. The part of plant used to induce callus is called explant which can be tissue, organ or part of a plant.

Organ culture is used to maintain or grow an entire organ or a part of organ in vitro. It preserves the structure and function of the organ for studies or transplantation.

Embryo culture involves isolating and growing embryos from seeds or ovules in a nutrient medium. It is used to bypass seed dormancy or rescue embryos from hybrid crosses that may not develop naturally.

Cell suspension culture involves growing dispersed cells or small cell clusters in a liquid medium under continuous shaking. This is used for large-scale production of cells, secondary metabolites, and studying cell biology..

Introduction
The process of culturing plant cells, tissues, organs and protoplast on artificial media under controlled and aseptic circumstances is known as ‘Plant Tissue Culture’. It is predicated on the idea of “Cellular totipotency”, which describes a cell’s capacity to develop into a whole plant in the right cultural environment. This characteristic of cells has broad applications in the manipulation of plant cells to enable fast plant multiplication and the in-vitro regeneration of whole plants following the genetic transformation. Around a century ago, the idea of tissue culture was first put forth, to grow entire plants in vitro from somatic cells (Haberlandt, 1902). The tissue culture system has advanced with the identification of different concentration, ratios of auxin and cytokinin, which are critical for the regeneration of adventitious roots and shoots (Skoog and Miller, 1957). Appropriate explant, or plant organ removed, including cotyledons, leaves, roots, shoot apices, nodal segments, hypocotyls, anthers, embryos, and seeds. The explants are thoroughly cleaned with sterile double-distilled water, surface sterilized with a disinfectant such as sodium hypochlorite or mercuric chloride and aseptically cultured in an artificial medium in test tubes/ flasks, jars, and Petri dishes. Several factors influence a plant’s capacity to regenerate, such as the presence of a plant growth regulator (Gerdakaneh et al., 2020), the explant type (Dhar and Joshi, 2005) and the basic medium’s composition (Sundararajan et al., 2017).
The most widely used media include major elements, carbohydrates, growth regulators (auxins, cytokinins etc.), microelements, vitamins, amino acids and several media compositions have been created specifically for plant tissue culture (Atkinson et al. 2012). While 2,4-dichloro phenoxy acetic acid (2,4-D) at concentrations of 0.5-4.0 mg/L typically induces callus, or a homogeneous mass of undifferentiated cells, auxins at concentrations ranging from 0.1 to 5.0 mg/L, favour rooting by cell elongation. Similarly, cytokinin’s induce fast cell division and the growth of shoot buds/shoots. Agaragar, agarose, and GelriteTM are examples of chemically inert, powdered gelling agents added to the culture medium to solidify it before it is autoclaved. After the medium has been added to the culture containers, they are autoclaved. Explants in the culture vessels are injected under aseptic circumstances in a laminar air-flow cabinet equipped with high-efficiency particulate air (HEPA) filters (pore size 0.2–0.3 mm). Explants that are placed in the right growth medium under the right circumstances dedifferentiate, or mature cells that have increased DNA/ RNA and protein production return to their meristematic state. Regrowth in the agar-gelled medium results in an amorphous mass of cells known as ‘callus’. Suspension culture, a method of cultivating cells in a liquid medium, produces a suspension of individual cells. Every three to four weeks, the developing tissue must be sub-cultured onto a new medium because an increase in cells or calli causes the medium to run out. In the end, the organogenesis pathway is utilized to create ordered structures like roots, branches, flower buds, etc. from these cells and tissues. The synthesis, preservation, and application of

5.1 Plant Breeder’s Rights (PBR) and Protection of Plant Varieties and Farmers’ Rights (PPV&FR) Act
5.1.1 Introduction
In the context of biotechnology and agricultural innovation, Plant Breeder’s Rights (PBRs) offer exclusive intellectual property protection to individuals or organizations that breed, develop, or discover new plant varieties. These rights ensure economic incentives for plant breeders while promoting innovation in seed development and agricultural productivity. In India, the unique and inclusive Protection of Plant Varieties and Farmers’ Rights Act (PPV&FR), 2001, balances breeders’ rights with recognition of farmers’ contributions, making it one of the most progressive legislations in the world.
5.1.2 Concept of Plant Breeder’s Rights (PBR)
Plant Breeder’s Rights refer to the exclusive rights granted to plant breeders to produce, sell, market, distribute, import or export their registered plant varieties. These rights are sui generis, meaning they are a special form of IP distinct from patents, developed to cater specifically to plant varieties.
Key Objectives of PBR
• Encourage investment in plant breeding and biotechnology.
• Ensure access to improved crop varieties.
• Prevent unauthorized commercial exploitation of novel varieties.
• Facilitate biodiversity conservation through formal registration systems.

The International Union for the Protection of New Varieties of Plants (UPOV) based in Geneva, Switzerland and established in 1961 is an intergovernmental organization to provide and promote an effective system of plant variety protection, with the aim of encouraging the development of new varieties of plants, for the benefit of society. For plant breeders' rights to be granted, the new variety must meet four criteria under the rules established by UPOV as discussed earlier also.

Abstract

The nutrition of tobacco plants relies upon the plant genotypic variation and soil characteristics which also influence the various microflora that inhabit the rhizosphere. The investigations were aimed to analyze the role of native and inoculated plant growth promoting bacteria on the uptake of major plant nutrients viz., nitrogen, phosphorus and potassium and also their supportive nature in reducing the infection caused by  Pythium aphanidermatum, the damping-off fungus of tobacco in nursery. Rhizosphere microorganisms include PGPR viz., Azotobacter, Azospirillum, Pseudomonas and Bacillus, AM fungi and Streptomyces. The addition of these microbes as bioinoculants in tobacco integrated nutrient and disease management resulted in better quality tobacco worth premium price.

In this chapter, a comprehensive list of all plants and trees mentioned in epic Ramayana has been furnished with salient details in order to establish their botanical identity and references of Sholka where it had been mentioned. Some of the plants and trees had been mentioned in multiple sholokas but the plant has been taken into account as a single reference to avoid repetition and to get correct count of the trees and plants in whole of the Epic (Amirthalingam, 2013, Arun Kumar, 2019; Balapure et al., 1987; Bast, 2022; Ghimire et.al, 2021; Mao and Dash, 2010; Prasad and Mishra, 1976).

The plant species mentioned in Ramayana were of different types having various usages. Considering their morphological characteristics, usages and other importance, they have been categorized broadly into the following heads.

1. Economically important plants (Timber value, product yielding etc. having commercial importance).

2. Edible (major and minor fruits etc.).

3. Culturally important plants (Plants used for plantation for shade, greening etc.).

4. Non-Edible Plants (Ornamental importance and other usages)

5. Medicinal Plants (having therapeutic uses).

6. Sacred Plants (having religious importance).

7. Others (having multipurpose uses).

Isolation of plasmid DNA from E. coli is a common routine in research laboratories. One can perform a widely-practiced procedure that involves alkaline lysis of cells. This protocol often referred to as a plasmid “mini-prep,” yields fairly clean DNA quickly and easily.

Procedure

Fill a microcentrifuge tube with saturated bacterial culture grown in LB broth + antibiotic. Spin tube in microcentrifuge for 1 minute, and make sure tubes are balanced in microcentrifuge. Dump supernatant and drain tube briefly on paper towel.

Repeat step 1 in the same tube, filling the tube again with more bacterial culture. The purpose of this step is to increase the starting volume of cells so that more plasmid DNA can be isolated per prep. Spin tube in microcentrifuge for 1 minute. Pour off supernatant and drain tube on paper towel.

Add 0.2 ml ice-cold Solution 1 to cell pellet and resuspend cells as much as possible using disposable transfer pipet.

Solution Preparation

1. Glycerol mix (1 L)

Weigh 25 grams (liquid weight) of glycerol and

Add dH₂O to 1 Liter (Autoclave).

2. Potassium mix (1 L)

170 mL of 1M KH₂PO₄ (monobasic),

720 mL of 1M K₂HPO₄ (dibasic), and

110 mL of dH₂O (Autoclave)

Principle

The principle is to extract plasmid DNA from bacterial cell suspensions and is based on the alkaline lysis procedure developed by Birnboim and Doly (Nucleic Acids Research 7:1513, 1979). It is a known fact that plasmids are much smaller super coiled DNA molecules than the much larger and less super coiled bacterial chromosomal DNA. This difference in topology enables selective precipitation of the chromosomal DNA and cellular proteins and separates them from plasmids and RNA molecules. The cells are lysed under alkaline conditions, which denature both nucleic acids and proteins, and when the solution is neutralized by the addition of Potassium Acetate, chromosomal DNA and proteins precipitate because it is impossible for them to renature correctly (they are so large). Plasmids renature correctly and stay in solution, effectively separating them from chromosomal DNA and proteins.

Principle

Apart from PCR for generating large amounts of DNA, the second method is to use bacteria to replicate the DNA and then purify the replicated DNA from the bacteria. Plasmid DNA is usually the DNA of interest for this method. A plasmid is a double stranded circular DNA molecule that will replicate in an organism. A typical plasmid contains four important features:

The plasmid must be circular, because bacteria generally will not replicate linear ? DNA.

The plasmid must contain a sequence that functions as an origin of replication (ori).

The plasmid must contain a selection mechanism that will force the bacteria to retain the DNA; the most common type of selection mechanism used in bacteria is a gene for resistance to an antibiotic such as ampicillin or tetracycline.

The plasmid must contain a region for the insertion of the experimental DNA. A generic plasmid exhibiting these features is shown at right.

An expression plasmid that allows expression of foreign DNA should have a strong promoter segment for transcription of inserted DNA.

Must have an effective ribosome binding site that will allow efficient translation of the transcribed RNA.

Must have high copy number, i.e., the host bacterium should have many copies (usually >100)

A TTO fills the important intermediary position of linking research institution with industry so that innovations and new ideas can reach market. To be able to facilitate that effectively, the institutions must have well-defined R&D policies that include Sponsored R&D, Collaborative R&D, and Grant-in-Aid R&D. These ensure that the research remains properly managed, regulatory compliance assured and aligned with institutional priorities. This chapter has discussed about key policies that can help in successful operation of TTO while aligning institution’s mission and interests Various core policies that help in successful technology transfer include:
• Intellectual property policy
• Technology transfer policy
• Revenue sharing policy
• Startup/Spinout/Venture creation policy
• Conflict of interest/Conflict of commitment policy
Each policy has specific objectives. These policies collectively ensure a clear and consistent framework for technology commercialization.
11.1 Intellectual property policy
An IP policy is a formal document that provides guidelines for managing IP by an organisation. A strong IP policy build trust by ensuring efficiency and transparency. Other benefits of IP policy includes creation of environment to encourage innovation and commercialisation of IP. It also lays foundation to set out the rules for public-private partnerships while addressing issues among various stakeholders and ensures compliance. IP Policy is not law, but a proposed course of action.

Pollination can be defined as the pre-fertilization event or process where pollen grains from anther are transferred to the stigma of a flower. Pollination is the process that helps to unite the male and female gametes and thus helps in fertilization. It can be broadly classified into two, cross-pollination and self-pollination.

1. Self-Pollination

Self-pollination is the process by transferring the pollen grains directly from anther into the stigma of the same flower. It is called as self-pollination. This form of pollination is common in hermaphrodite or dioecious plants which have both male and female sexual organs on the same flower. In self-pollinating flowers, the anthers and stigma are of similar lengths to facilitate the transfer of pollen. Self – pollination can be further divided into two types:

Principle

Monomeric acrylamide (which is neurotoxic) is polymerised in the presence of free radicals to form polyacrylamide. The free radicles are provided by ammonium persulphate and stabilised by TEMED (N’N’N’N’-tetramethylethylene-diamine). The chains of polyacrylamide are cross-linked by the addition of methylenebisacrylamide to form a gel whose porosity is determined by the length of chains and the degree of crosslinking. The chain length is proportional to the acrylamide concentration: usually between 3.5 and 20%. Cross-linking bisacrylamide is usually added at a ratio of 2g BIS 38g acrylamide.

Polyacrylamide

Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%. Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Because oxygen inhibits the polymer- ization process, they must be poured between glass plates (or cylinders).

Denaturing polyacrylamide/urea gels in TBE buffer

Prepare 20ml of a 5% polyacrylamide gel containing 7M urea by adding:

47.5% acrylamide:

2.5% bis-acrylamide solution 2ml

10M urea 14ml

10X TBE buffer 2ml

10% freshly prepared ammonium persulfate 0.2ml

deionized water 1.8ml

PCR Principles and Procedure

Polymerase chain reaction (PCR) is a technique that allows the generation of large amounts of a single DNA sequence from a mixture of sequences; the fragment generated can be designed to contain specific starting and ending positions based on the needs of the experiment. PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.

A basic PCR set up requires several components and reagents. These components include:

Introduction

This chapter gives first hand information on PCR for beginners. PCR is used to amplify a short, well-defined part of a DNA strand. This can be a single gene, or just a part of a gene. As opposed to living organisms, the PCR process can copy only short DNA fragments; usually up to 10 kb (kb stands for kilo base pairs). Certain methods can copy fragments up to 40 kb in size, which is still much less than the chromosomal DNA of a eukaryotic cell-for example, a human cell contains about three billion base pairs (Kary Mullis, 1983).